Quantitative standard for mass spectrometry of proteins

ABSTRACT

The present invention provides a method of determining the absolute amount of a target polypeptide in a sample, said method comprising the following steps: (a) adding (aa) a fusion polypeptide to said sample, said fusion polypeptide comprising (i) at least one tag sequence and (ii) a subsequence of the target polypeptide; and (ab) a known absolute amount of a tag polypeptide comprising or consisting of said tag sequence according to (aa) to said sample, wherein said fusion polypeptide on the one hand is mass-altered as compared to said target polypeptide and said tag polypeptide on the other hand, for example, said fusion polypeptide on the one hand and said target polypeptide and said tag polypeptide on the other hand are differently isotope labeled; (b) performing proteolytic digestion of the mixture obtained in step (a); (c) subjecting the result of proteolytic digestion of step (b), optionally after chromatography, to mass spectrometric analysis; and (d) determining the absolute amount of said target polypeptide from (i) the peak intensities in the mass spectrum acquired in step (c) of said fusion polypeptide, said tag polypeptide and said target polypeptide and (ii) said known absolute amount of said tag polypeptide. 
     Furthermore provided is a fusion polypeptide for the quantification of a target polypeptide by mass spectroscopy, wherein: said fusion polypeptide consists of 35 to 455 amino acid residues and comprises (i) a target region, which is a fragment of the target polypeptide, and (ii) a tag region, which is not a fragment of the target polypeptide, said target region consists of 15 to 205 amino acid residues and comprises at least two signature regions; said tag region consists of 20 to 250 amino acid residues and comprises at least two signature regions; and each signature region has the structure Y-Z-X 4-28 -Y-Z, wherein all Y:s are selected from one of (i)-(iv), wherein (i) is R or K, (ii) is Y, F, W or L, (iii) is E and (iv) is D and each X and each Z are independently any amino acid residue, provided that the Z:s are not P if the Y:s are selected from (i)-(iii); and each signature region comprises at least one amino acid residue comprising a heavy isotope.

FIELD OF THE INVENTION

This invention relates to a method of determining the absolute amount of a target polypeptide in a sample, said method comprising the following steps: (a) adding (aa) a fusion polypeptide to said sample, said fusion polypeptide comprising (i) at least one tag sequence and (ii) a subsequence of the target polypeptide; and (ab) a known absolute amount of a tag polypeptide comprising or consisting of said tag sequence according to (aa) to said sample, wherein said fusion polypeptide on the one hand is mass-altered as compared to said target polypeptide and said tag polypeptide on the other hand, for example, said fusion polypeptide on the one hand and said target polypeptide and said tag polypeptide on the other hand are differently isotope labeled; (b) performing a proteolytic digestion of the mixture obtained in step (a); (c) subjecting the result of the proteolytic digestion of step (b), optionally after chromatography, to mass spectrometric analysis; and (d) determining the absolute amount of said target polypeptide from (i) the peak intensities in the mass spectrum acquired in step (c) of said fusion polypeptide, said tag polypeptide and said target polypeptide and (ii) said known absolute amount of said tag polypeptide.

BACKGROUND OF THE INVENTION

In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Mass spectrometry (MS)-based proteomics has become a method of choice to study proteins in a global manner (1-3). Mass spectrometry is not inherently quantitative but methods have been developed to address this limitation to a certain extent. Most of them are based on stable isotopes and introduce a mass shifted version of the peptides of interest, which are then quantified by their ‘heavy’ to ‘light’ ratio. Stable isotope labeling is either accomplished by chemical addition of labeled reagents, enzymatic isotope labeling or metabolic labeling (4-6). Generally, these approaches are used to obtain relative quantitative information on proteome expression levels in a light and a heavy labeled sample. For example, stable isotope labeling by amino acids in cell culture SILAC (7, 8) is performed by metabolic incorporation of differently labeled, such as light or heavy labeled amino acids into the proteome. Labeled proteomes can also be used as internal standards for determining protein levels of a cell or tissue proteome of interest, such as in the spike-in SILAC approach (9).

Absolute quantification is technically more challenging than relative quantification and could so far only be performed accurately for a single or a small number of proteins at a time (10). Typical applications of absolute quantifications are the determination of cellular copy numbers of proteins (important for systems biology) or the concentration of biomarkers in body fluids (important for medical applications). Furthermore, any precise method of absolute quantification, when performed in more than one sample, also yields the relative amounts of the protein between these samples.

Several methods for absolute quantification have emerged over the last years including AQUA (11), QConCAT (12, 13), PSAQ (14), absolute SILAC (15) and FlexiQuant (16). They all quantify the endogenous protein of interest by the heavy to light ratios to a defined amount of the labeled counterpart spiked into the sample and are primarily distinguished from each other by either spiking in heavy labeled peptides or heavy labeled full length proteins. The AQUA strategy uses proteotypic peptides (17) which are chemically synthesized with heavy isotopes and spiked in after sample preparation. AQUA peptides are commercially available but expensive, especially when many peptides or proteins need to be quantified (see, for example, Kettenbach et al., Nat. Protoc. 2011, 6:175-86). Moreover, the AQUA strategy suffers from quantification uncertainties that are introduced due to spiking in of the peptide standard after sample preparation and enzymatic proteolysis, which is a late stage in the workflow. Furthermore, any losses of the peptides—for example during storage—would directly influence quantification results. The QconCAT approach is based on artificial proteins that are a concatamers of proteotypic peptides. This artificial protein is recombinantly expressed in Escherichia coli and spiked into the sample before proteolysis. QconCAT allows production of labeled peptides, but does not correct any bias arising from protein fractionation effects or digestion efficiency. The PSAQ, absolute SILAC and FlexiQuant approaches try to address these limitations by metabolically labeling full length proteins by heavy versions of the amino acids arginine and lysine. PSAQ and FlexiQuant synthesize full-length proteins in vitro in wheat germ extracts or in bacterial cell extract, respectively, whereas absolute SILAC was described with recombinant protein expression in E. coli. The protein standard is added at an early stage, such as directly to cell lysate. Consequently, sample fractionation can be performed in parallel and the SILAC protein is digested together with the proteome under investigation. However, these advantages come at the cost of having to produce full length proteins, which limits throughput and generally restricts these methods to soluble proteins.

Accordingly, there is an unmet need for improved or alternative means and methods of mass spectrometry-based absolute quantitation of peptides and polypeptides.

DETAILED DESCRIPTION

The present invention provides a method of determining the absolute amount of a target polypeptide in a sample, said method comprising the following steps: (a) adding (aa) a fusion polypeptide to said sample, said fusion polypeptide comprising (i) at least one tag sequence and (ii) a subsequence of the target polypeptide; and (ab) a known absolute amount of a tag polypeptide comprising or consisting of said tag sequence according to (aa) to said sample, wherein said fusion polypeptide on the one hand is mass-altered as compared to said target polypeptide and said tag polypeptide on the other hand, for example, said fusion polypeptide on the one hand and said target polypeptide and said tag polypeptide on the other hand are differently isotope labeled; (b) performing proteolytic digestion of the mixture obtained in step (a); (c) subjecting the result of proteolytic digestion of step (b), optionally after chromatography, to mass spectrometric analysis; and (d) determining the absolute amount of said target polypeptide from (i) the peak intensities in the mass spectrum acquired in step (c) of said fusion polypeptide, said tag polypeptide and said target polypeptide and (ii) said known absolute amount of said tag polypeptide.

The term “absolute amount” has its usual meaning and is to be held distinct from relative amounts, i.e. ratios, as they are commonly determined in expression analysis, be it by mRNA expression profiling or proteomics methods. In particular, it is understood that the term “absolute amount” refers to the copy number or the amount of substance of a given protein or polypeptide in, for example, a cell, or the amount in a defined volume, or in a sample such as ng/mL of a body fluid such as urine or plasma. In other words, said absolute amount may be expressed in terms of a concentration, a mass or amount of substance (in moles or number of molecules).

The term “polypeptide” is well established in the art and refers to a polycondensate of amino acids, preferably of the 20 standard amino acids. It is understood that the term “polypeptide” as used herein embraces also peptides, wherein peptides have a minimal length of two amino acids. On the other hand, the term “polypeptide” includes proteins, at least to the extent such proteins consist of a single chain. Proteins in turn may also comprise more than one polypeptide chain.

It is understood that the methods according to the invention are equally suitable to determine the absolute amounts of proteins, also to the extent proteins comprise more than one polypeptide chain. In such a case, and assuming the molar ratios of the polypeptide chains comprised in the protein are known, it may be sufficient to determine the absolute amount of one polypeptide comprised in the protein of interest. Alternatively, the absolute amount of more than one or all polypeptides comprised in the protein of interest may be determined by the methods according to the invention.

A “fusion polypeptide” according to the invention is a polypeptide which comprises at least two segments of different origin. More specifically, a fusion polypeptide according to the invention requires presence of a tag amino acid sequence and a subsequence of the target polypeptide comprised or suspected to be comprised in the recited sample. It is deliberately envisaged that more than one tag amino acid sequence is present. This is the subject of preferred embodiments discussed further below. Furthermore, this is exemplified in the enclosed examples and depicted in FIG. 1. Preferred embodiments of the fusion polypeptides are described further below and include protein epitope signature tags (PrESTs). It is preferred that said tag sequence is chosen such that proteolytic digestion of the target proteome on the one hand and of the tag sequence on the other hand yield two disjunct sets of peptides or at least two sets of peptides which overlap by less than 25%, less than 10%, less than 5%, less than 2% or less than 1%. A “target proteome” is typically a proteome originating from a single species. A target proteome comprises said target polypeptides. A preferred proteome is a human proteome. If more than one tag sequence is present, it is understood that the tag sequences are different from each other. In particular, the set of peptides obtained by proteolytic digestion of a first tag sequence present in said fusion polypeptide and the set of peptides obtained by proteolytic digestion of a second tag sequence (and also any further tag sequence) present in said fusion polypeptide are disjunct, i.e., they do not a share a peptide of same sequence. Whenever reference is herein made to disjunct sets of peptides obtained by proteolytic digestion, it is understood that the sets of peptides are in particular disjunct as regards peptides of or above a minimal length, said minimal length being at least 4, 5, 6, 7, 8 or 9 amino acids.

The term “subsequence” in its broadest form refers to any partial sequence of a target polypeptide to be detected and furthermore includes the entire sequence of said target polypeptide. In a preferred embodiment, said subsequence is a partial sequence of the target polypeptide, the entire sequence of said target polypeptide being excluded. Preferred length ranges of said subsequence are discussed further below.

The term “isotope” refers to two or more nuclides with the same number of protons (atomic number) but different numbers of neutrons. Such difference in mass number provides for different peak positions of an isotope labeled compound or fragment on the one hand and its unlabeled counterpart on the other hand in a mass spectrum. Preferred isotopes are deuterium, 13C and 15N.

The term “labeled” refers to a frequency of isotopes which deviates from the naturally occurring frequency. In preferred embodiments, the term “isotope labeled” refers to a compound, moiety, fragment or molecule which, to the extent atoms with the same atomic number are considered, exclusively contains a given isotope. For example, a preferred isotope labeled lysine has 13C nuclides at all carbon positions. In preferred embodiments, one or more specific amino acids, such as all lysines and/or all arginines, are isotope labeled. Suitable isotope labeled amino acid residues are listed further below.

The term “differently labeled” or “differently isotope labeled” as used herein refers to a plurality of labeling schemes. In particular, it is sufficient for two polypeptides to be differently labeled, if one of them is labeled and the other one is not. Equally envisaged is that one of the polypeptides is isotope labeled in one specific way, whereas the other polypeptide is isotope labeled as well, but in a different way, the consequence being that both polypeptides do not exhibit the naturally occurring frequency of isotopes and can be distinguished in the mass spectrum. It is understood that “differently isotope labeled” according to the invention is such that, upon proteolytic digestion, (i) at least a first peptide is formed from the target polypeptide and at least a second peptide is formed from the subsequence thereof as comprised in the fusion polypeptide such that the first and second peptide are identical in sequence but differ in their mass, and (ii) at least a third peptide is formed from the tag polypeptide and at least a fourth peptide is formed from the tag sequence as comprised in the fusion polypeptide such that the third and fourth peptide are identical in sequence but differ in their mass. This can be achieved, for example, by the labeled polypeptides comprising internal labels, preferably each occurrence of one or more given amino acids being labeled, said given amino acids being preferably those which are comprised in the cleavage site recognized by the enzyme used for proteolytic digestion. Such preferred amino acids are, as described elsewhere herein, lysine and/or arginine. Taken together, it is preferred that said fusion polypeptide on the one hand and said target polypeptide and said tag polypeptide on the other hand are differently internally isotope labeled. The term “internal” as used herein in relation to labels is understood to distinguish from terminal labels.

Generally speaking, whenever reference is made to “differently labeled” or “differently isotope labeled” in the present disclosure, it is understood that these terms relate to a preferred embodiment. More generally, any means of mass-alteration including, though not confined to isotope labeling is envisaged. The terms “mass-alteration” and “mass-altered” as used herein refer to all those means and methods which provide for peptides (or polypeptides) obtained from different sources and identical in sequence to differ with regard to their mass. Isotope labeling is one preferred means of achieving this goal. An alternative method known in the art is the use of isobaric tags for relative and absolute quantitation (iTRAQ). This method uses isotope-coded covalent tags; see, for example, Ross et al., Mol. Cell. Proteomics 3, 1154-69, 2004. Preferably, iTRAQ is based on a covalent labeling of the N-terminus and sidechain amines of peptides and polypeptides. Suitable agents are known in the art, examples of which include agents referred to as 4-plex and 8-plex. If it is stated herein that an entity A is mass-altered as compared to an entity B, it is understood that either entity A or entity B deviates from the naturally occurring form, for example by different isotope labeling or owing to the presence covalent tags in the sense of iTRAQ.

Turning to the requirement as recited in the main embodiment that “at least said fusion polypeptide on the one hand and said target polypeptide and said tag polypeptide on the other hand are differently isotope labeled”, it is noted that said target polypeptide and said tag polypeptide may be isotope labeled in the same way or according to different labeling patterns, or, if said fusion polypeptide is isotope labeled, both may be unlabeled. More specifically, at least the following labeling schemes are embraced. (1) Said fusion polypeptide is isotope labeled, and both said target polypeptide and said tag polypeptide are not isotope labeled, (2) said target polypeptide and said tag polypeptide are isotope labeled, and said fusion polypeptide is not isotope labeled, wherein target polypeptide and tag polypeptide are isotope labeled in the same way or according to different labeling patterns, (3) a polypeptide selected from target polypeptide, fusion polypeptide and tag polypeptide is not isotope labeled or isotope labeled according to a first pattern, a second polypeptide chosen from the same group is isotope labeled according to a second pattern, and the remaining polypeptide from the group is isotope labeled according to a third pattern. The three patterns (or two patterns in case one of the polypeptides is not isotope labeled) according to labeling scheme (3) may be implemented, for example, by using two or three isotope labeled forms of one or more given amino acids, said two or three isotope labeled forms differing in the total mass. An exemplary labeling scheme according to (3) is as follows: the target polypeptide is not isotope labeled, the fusion polypeptide is isotope labeled (“heavy weight” form), and the tag polypeptide is isotope labeled according to a different pattern such that it is provided, for example, either in a “middle weight” or an “extra heavy weight” form. Such a labeling scheme may be particularly preferred if it is suspected that a proteolytic product of the tag polypeptide could also be derived from the digestion of the sample, e.g. if the sample is human and the tag is a human protein or a domain or segment thereof.

The term “labeling scheme” as used herein distinguishes between different polypeptides. For a given labeling scheme, a class of polypeptides (classes being target polypeptides, tag polypeptides, and fusion polypeptides) is labeled in the same way, for example by incorporation of a 13C labeled lysine at all positions where a lysine occurs. A labeling scheme provides for different classes being differently labeled. On the other hand, the term “labeling pattern” distinguishes between differently labeled forms of a given peptide. For example, a specific polypeptide may be labeled by replacing all occurrences of lysine with 13C labeled lysine or by replacing all positions of arginine with 13C 15N labeled arginine, thereby rendering the labeling patterns differently.

Various means for isotope labeling are at the skilled person's disposal and include chemical addition of labeled reagents, enzymatic isotope labeling or metabolic labeling (4-6).

According to the invention it is preferred that the isotope labeling is introduced by metabolic labeling. In other words, the polypeptides to be used in the methods according to the invention, to the extent they are required to be labeled, are preferably obtained by means of production in biological systems, such as cell-free as well as cellular systems. For example, a host cell may be used which is auxotrophic for lysine and/or arginine, wherein at the same time isotope labeled lysine and/or arginine is provided in the growth medium. A preferred means of metabolic isotope labeling is stable isotope labeling with amino acids in cell culture (SILAC). SILAC procedures are known in the art and described in the background section herein above as well as in the references cited in relation thereto which are herewith incorporated by reference. As mentioned above, to the extent isotope labeling makes use of isotopes with higher mass numbers, the labeled form is commonly referred to as “heavy” form, whereas the naturally occurring counterpart or the counterpart which is free or essentially free of the heavy isotope under consideration is commonly referred to as “light” form.

The recited “known absolute amount of a tag polypeptide” may be determined with methods established in the art. A preferred method is amino acid analysis. Amino acid analysis is typically provided as a service by a variety of companies. The method preferably includes the total hydrolysis of a given sample, the chemical derivatization of the obtained free amino acids, the separation of the derivatized amino acids, for example by reversed phase HPLC, and the subsequent interpretation of the result. The method is described in more detail in, for example, in Moore and Stein, J. Biol. Chem. 176, 367-388 (1948) as well as in Moore and Stein, J. Biol. Chem. 176, 337-365 (1948).

The methods according to the invention require, on the one hand, that a first subsequence of the fusion polypeptide is identical to a subsequence of the target protein, and on the other hand, that a second subsequence of the fusion polypeptide is identical to the tag polypeptide. Furthermore, even though the amino acid sequences are identical, the masses of the first subsequence of the fusion polypeptide and its counterpart in the target polypeptide need to be distinct. Likewise, the masses of the second sequence of the fusion polypeptide and the tag polypeptide also need to be distinct. This may be achieved by the labeling schemes described above. This allows for quantitative comparisons to be made between the tag sequence within the fusion polypeptide and the tag polypeptide as well as between said subsequence comprised in said fusion polypeptide and the target polypeptide polypeptide.

Step (b) provides for proteolytic digestion that, as is well established in the art, gives rise to fragments which can conveniently be handled in mass spectroscopy. Preferred enzymes to be used for proteolytic digestion are described further below. It is preferred that said proteolytic digestion is specific, i.e., that cleavage occurs at all cleavage sites of the enzyme used. On the other hand, and as described herein, the methods of the present invention provide for the avoidance of bias introduced by incomplete digestion.

Subsequent to proteolytic digestion, mass spectrometry analysis is performed. Ionized peptide molecules are transferred into the vacuum systems of the mass spectrometer. In a preferred mode of operation, widely known to the practitioners of the art, the mass spectrometer is operated so as to perform a mass spectrometric scan that records a mass spectrum of the peptides entering the instrument at that time. Quantification is based on the peaks present in this mass spectrometric (or MS) scan. The enclosed examples provide a more detailed account of suitable modes of operation of the mass spectrometer. Depending on the nature of the samples to be analyzed, the polypeptides suspected to be comprised in the sample and the available instrumentation, the skilled person can choose suitable modes of operation.

Given that proteolytic digestion is performed, the tag polypeptide comprising said tag sequence according to (aa) or a tag polypeptide consisting of said tag sequence according to (aa) may be used interchangeably. Preferably, in either case the same one or more tag fragments will be yielded during proteolytic digestion.

Prior to performing mass spectrometry analysis, the result of proteolytic digestion may be subjected to chromatography as is established in the art. Preferred means of chromatography are liquid chromatography (LC). In a preferred mode of operation, the peptide mixture is injected onto a liquid chromatographic column, separated by a gradient of organic solvent lasting several minutes or several hours and on-line electrosprayed.

Step (d) combines the information obtained in the mass spectrum (which can be viewed as relative intensities) with the known absolute amount of the tag polypeptide in order to determine absolute amounts, in particular the absolute amount of the target polypeptide comprised in the sample. To explain further, and using the terminology of first to fourth peptides introduced herein above, the absolute amount of a given target polypeptide may be determined, for example, as follows. Ratios of amounts of substance are identical to ratios of intensities in the MS spectrum of the corresponding peaks. Using the numbers from 1 to 4 as short hand designations of first to fourth peptide, the following applies. The amount of substance of the fourth peptide (proteolytic fragment derived from the tag sequence as comprised in the fusion polypeptide) N(4) can be determined according to N(4)=N(3) times I(4)/I(3). N(3) is the known absolute amount of the tag polypeptide. I(3) and I(4) are the corresponding peak intensities. Given the definition of the fusion polypeptide, N(2)=N(4) applies, i.e. the amounts of substance of the peptides formed from either part of the fusion polypeptide are identical. The amount of substance of the target polypeptide N(1) can then be determined as follows: N(1)=N(2) times I(1)/I(2). Making use of N(2)=N(4) and N(4)=N(3) times I(4)/I(3), it follows that N(1)=N(3) [I(1) I(4)/I(2) I(3)] which permits absolute quantitation of the target polypeptides based on peak intensities I(1) to I(4) and the known absolute amount of the tag polypeptide N(3). Note that in practice the ratios are usually determined as the mean of the ratios of several peptide intensities; i.e. more than one peptide pair covering the tag sequence and the target polypeptide sequence.

The methods according to the invention make use of specific labeling schemes of three distinct species, the labeling schemes being described above. A key feature of the methods of the invention is the use of fusion polypeptides, said fusion polypeptides containing at least one generic sequence, also referred to as “tag sequence” herein. The concomitant provision of a tag polypeptide as defined above in a known absolute amount permits calibration in a manner which advantageously is independent of the actual polypeptide to be quantitatively determined.

Deviating from a variety of prior art methods as discussed above, the methods of the present invention provide for early adding of the standard (in case of the main embodiment said known absolute amount of a tag polypeptide) in the entire workflow. As a consequence, downstream steps including proteolytic digestion and optionally chromatography is equally applied to both the standard and the constituents of the sample to be analyzed. Any variation in efficiency or performance of, for example proteolytic digestion, will equally affect all constituents of the mixture obtained in step (a), thereby avoiding any bias that could arise therefrom. In a preferred embodiment, no protein size-based methods such as size exclusion chromatography is used after said adding.

It is well known to practitioners of proteomics that accurate quantification of proteins of very low abundance proteins is challenging. However, the accuracy of quantification of the fusion protein standard itself does not depend on the cellular abundance or other attributes of the polypeptide to be determined, noting that the same amount of fusion polypeptide is preferably used in each instance of the methods according to the invention. Also, the purity of a composition comprising said fusion polypeptide to be added has no impact because the methods specifically determine the amount of the fusion polypeptide and not of total protein.

As discussed in more detail in the examples enclosed herewith, the methods according to the present invention provide for significantly improved accuracy in quantitative determination of cellular protein expression levels. Further advantages of the method are that it typically results in several quantifiable peptides for each fusion polypeptide, both for the accurate quantification of the standard and for the target polypeptide to be absolutely quantified. Furthermore, production of the standard can be streamlined because protein expression can be performed in a standard system (such as E. coli) and because a large number of fusion polypeptides can be produced under similar conditions as they only differ by a relatively short unique sequence in the preferred embodiment.

In a second aspect, the present invention provides a method of creating a quantitative standard, said method comprising the following steps: (a) providing a plurality of fusion polypeptides, each of said fusion polypeptides comprising (i) at least one tag sequence and (ii) a subsequence of a target polypeptide to be quantitatively determined, wherein all fusion polypeptides share at least one tag sequence, thereby obtaining the standard; (b) determining the absolute amounts of said fusion polypeptides by (ba) adding to one of said fusion polypeptides at a time a known amount of a tag polypeptide comprising or consisting of the tag sequence shared among the fusion polypeptides according to (a), wherein said fusion polypeptide is mass-altered as compared to said tag polypeptide, for example, said fusion polypeptide and said tag polypeptide are differently isotope labeled, (bb) performing proteolytic digestion of the mixture of one fusion polypeptide and said tag polypeptide obtained in step (ba); (bc) subjecting the result of proteolytic digestion of step (bb), optionally after chromatography, to mass spectrometric analysis; and (bd) determining the absolute amount of said one fusion polypeptide from (i) the peak intensities in the mass spectrum of fusion polypeptide and tag polypeptide and (ii) said known amount of said tag polypeptide, thereby obtaining the absolute amount of one of said fusion polypeptides at a time.

While the second aspect provides for the option of multiplexing as discussed further below, it is of note that said second aspect is not confined to the use of a plurality of fusion polypeptides. Accordingly, the present invention also provides a method of creating a quantitative standard, said method comprising the following steps: (a) providing one fusion polypeptide, the one fusion polypeptide comprising (i) at least one tag sequence and (ii) a subsequence of a target polypeptide to be quantitatively determined, thereby obtaining the standard; (b) determining the absolute amount of said fusion polypeptide by (ba) adding to the one fusion polypeptide a known amount of a tag polypeptide comprising or consisting of the tag sequence comprised in the one fusion polypeptide according to (a) wherein said fusion polypeptide is mass-altered as compared to said tag polypeptide, for example, said fusion polypeptide and said tag polypeptide are differently isotope labeled, (bb) performing proteolytic digestion of the mixture of one fusion polypeptide and said tag polypeptide obtained in step (ba); (bc) subjecting of the result of proteolytic digestion of step (bb), optionally after chromatography, to mass spectrometric analysis; and (bd) determining the absolute amount of said one fusion polypeptide from (i) the peak intensities in the mass spectrum of fusion polypeptide and tag polypeptide and (ii) said known amount of said tag polypeptide, thereby obtaining the absolute amount of the one fusion polypeptide.

In other words, part of a fusion polypeptide preparation is combined with a known amount of a tag polypeptide, wherein the fusion polypeptide is mass-altered as compared to the tag polypeptide. This binary mixture is subjected to proteolytic digestion, mass spectrometric analysis and quantitation to provide the absolute amount of the fusion polypeptides part, from which amount the exact concentration of the fusion polypeptide in the preparation can be calculated. Thus, a quantitative standard of a single fusion polypeptide has been provided. Then, at least part of the quantitative standard is added to the sample to be analyzed, after which proteolytic digestion of the obtained mixture is performed. The result of proteolytic digestion is subjected to to mass spectrometric analysis, optionally after chromatography. The absolute amount of the target polypeptide is then determined from (i) the peak intensities in the mass spectrum of the fusion polypeptide and the target polypeptide and (ii) the known absolute amounts of the fusion polypeptide, wherein said fusion polypeptide is mass-altered as compared to said target polypeptide.

Therefore, it is understood that said second aspect, in a more concise form covering both the use of one fusion polypeptide and a plurality thereof, relates to a method of creating a quantitative standard, said method comprising the following steps: (a) providing one or a plurality of fusion polypeptides, the one fusion polypeptide or each of said fusion polypeptides, respectively, comprising (i) at least one tag sequence and (ii) a subsequence of a target polypeptide to be quantitatively determined, wherein, to the extent said plurality of fusion polypeptides is provided, all fusion polypeptides share at least one tag sequence, thereby obtaining the standard; (b) determining the absolute amounts of said fusion polypeptide(s) by (ba) adding to the one fusion polypeptide or to one of said fusion polypeptides at a time, respectively, a known amount of a tag polypeptide comprising or consisting of the tag sequence comprised in the one fusion polypeptide or shared among the fusion polypeptides, respectively, according to (a), wherein said fusion polypeptide is mass-altered as compared to said tag polypeptide, for example, said fusion polypeptide and said tag polypeptide are differently isotope labeled, (bb) performing proteolytic digestion of the mixture of one fusion polypeptide and said tag polypeptide obtained in step (ba); (bc) subjecting of the result of proteolytic digestion of step (bb), optionally after chromatography, to mass spectrometric analysis; and (bd) determining the absolute amount of said one fusion polypeptide from (i) the peak intensities in the mass spectrum of fusion polypeptide and tag polypeptide and (ii) said known amount of said tag polypeptide, thereby obtaining the absolute amount of the one fusion polypeptide or of one of said plurality of fusion polypeptides at a time, respectively.

Related thereto, the present invention in a third aspect provides a method of determining the absolute amount of one or more target polypeptides in a sample, said method comprising the following steps: (a) optionally performing the method according to the second aspect; (b) adding the quantitative standard as defined in the second aspect to said sample; (c) performing proteolytic digestion of the mixture obtained in step (b); (d) subjecting the result of proteolytic digestion of step (c), optionally after chromatography, to mass spectrometric analysis; and (e) determining the absolute amounts of the target polypeptide(s) from (i) the peak intensities in the mass spectrum acquired in step (d) of fusion polypeptide(s) and target polypeptides and (ii) the known absolute amount(s) of said fusion polypeptide(s), wherein said fusion polypeptide(s) is/are mass-altered as compared to said target polypeptide(s), for example, said one or more target polypeptide(s) is/are differently isotope labeled as compared to said fusion polypeptide(s).

While the main embodiment provides for absolute quantitation of one polypeptide from a single mass experiment, the second and third aspects of the present invention relate to (i) preparation and quantitation of a standard and (ii) use of this standard in the quantitation of one or more of a plurality of polypeptides comprised in a sample. Importantly, such an approach is amenable to multiplexing. In other words, not only one, but also a plurality of polypeptides comprised in a sample can be concomitantly determined in a quantitative manner.

According to the second aspect, one or a plurality of fusion polypeptides is provided. According to step (b) of the second aspect, one fusion polypeptide at the time is combined with a known amount of a tag polypeptide. This binary mixture is subjected to proteolytic digestion, mass spectrometric analysis and quantitation to provide the absolute amount of one of said fusion polypeptides at a time. By performing step (b) of the second aspect for the one, more or all of the fusion polypeptides comprised in the standard, the standard is quantitatively characterized and can be used in a method in accordance with the third aspect of the present invention. The method of the second aspect provides in step (a) for the physical manufacture of the quantitative standard, and in step (b) for its characterization in terms of absolute amounts of the constituent fusion polypeptide(s). Preferred quantitative standards are also referred to as “PrEST master mix” herein.

A method according to the third aspect may, according to step (a), incorporate the method of creating a quantitative standard according to the second aspect of the invention in its entirety. Alternatively, step (a) may be omitted. In that case, it is understood that the quantitative standard to be added according to step (b) is characterized in accordance with step (b) of the second aspect.

Accordingly, in one embodiment, the internal standard (i.e. the fusion polypeptide) is thus quantified in a first step using an internal standard of the internal standard (i.e. the tag polypeptide), and a target protein in a sample is quantified in a subsequent second step using the quantified internal standard (i.e. the fusion polypeptide quantified in the first step). In this embodiment, the first step may be carried out at one site, such as at the premises of the company providing quantified fusion polypeptides, while the second step is carried out at another site, such as in a lab where proteins in biological samples are quantified for diagnostic purposes.

As recited in the third aspect, said one or more target polypeptides are mass-altered, preferably differently isotope labeled as compared to said fusion polypeptides. In other words, and in those cases where said fusion polypeptides are not isotope labeled, it is necessary to prepare a sample wherein the one or more target polypeptides comprised in the sample are isotope labeled. On the other hand, a requirement to prepare an isotope labeled sample does not arise for those embodiments falling under the third aspect where said fusion polypeptides are isotope labeled.

In a preferred embodiment, more than one fusion polypeptide comprising different subsequences of a target polypeptide in said sample are used. According to this embodiment, more than one fusion polypeptide is used in the quantitation of one given target polypeptide. This aspect is further described in the examples enclosed herewith and provides for improved accuracy and statistical significance.

In a further preferred embodiment, one or two tags are present in said fusion polypeptides, said tag(s) being selected from a purification tag and a solubility tag. This embodiment embraces the concomitant presence of two different tags. Preferred embodiments of either tag are described further below. It is understood that the solubility tag is preferably used as a quantitation tag (“tag sequence”) in accordance with the methods of the present invention.

In a further preferred embodiment of the methods of determining absolute amounts according to the invention, said sample comprises cells and/or body fluids. Said cells may be of various types or of a single type. Moreover, the cells may be embedded in one or more tissues. To the extent human cells are envisaged, it is preferred that such human cell is not obtained from a human embryo, in particular not via methods entailing destruction of a human embryo. On the other hand, human embryonic stem cells are at the skilled person's disposal. Accordingly, the present invention may be worked with human embryonic stem cells without any need to use or destroy a human embryo. The sample may comprise one or more body fluids, said body fluids preferably being selected from blood, blood serum, blood plasma, breast milk, cerebrospinal fluid, mucus, peritoneal fluid, pleural fluid, saliva, semen, sweat, tears, vaginal secretion and urine.

In a further preferred embodiment, said adding is effected prior to proteolytic digestion of the polypeptides. This embodiment relates to those cases where the sample to be analyzed comprises or consists of cells. Said adding refers to the addition of a fusion polypeptide and a tag polypeptide according to the main embodiment, or to adding the quantitative standard according to the third aspect of the invention. In either case, the early adding according to this embodiment provides for the methods to account for any bias possibly introduced by sample preparation and processing, in particular by the enzymatic digestion step. This is a further advantage as compared to those prior art methods which require a late spiking-in of the standard during the workflow.

In a further preferred embodiment, between two and 500 fusion polypeptides are used. As stated above, the second and third aspect of the invention provide for multiplexing. Preferred numbers of fusion polypeptides to be used in each instance of the method are between 2 and 200, such as between 2 and 100, including any integer value embraced by these lower and upper limits such as 50 fusion polypeptides. The examples enclosed herewith provide an account of excellent performance when using 43 fusion polypeptides.

In a further preferred embodiment, a solubility tag is present in each of said fusion polypeptides. A preferred solubility tag consists of the sequence of SEQ ID NO: 1. The sequence of SEQ ID NO: 1 is particularly advantageous in that the sequences obtained by tryptic digestion of the human proteome on the one hand and of the sequence of SEQ ID NO: 1 on the other hand are disjunct. In other words, a tryptic digestion of the sequence of SEQ ID NO: 1 yields peptides none of which is obtained from a tryptic digestion of the human proteome. The same applies at least for the majority of peptides obtained from the sequence of SEQ ID NO: 1 when the other preferred enzymes as disclosed herein are used for proteolytic digestion.

In a further preferred embodiment said subsequence of a polypeptide (a) consists of 15 to 205 amino acids; (b) comprises a proteotypic peptide; and/or (c) is selected to have minimal sequence identity to other proteins, excludes signal peptides and/or excludes sequences from transmembrane spanning regions. The subsequence recited in this embodiment is the subsequence of a target polypeptide as comprised in the fusion polypeptide according to the present invention. Feature (a) provides for a preferred length range of said subsequence. Further preferred lengths and length ranges are disclosed herein, in particular in the description of the fourth aspect of the invention. Such disclosure applies mutatis mutandis to the present preferred embodiment. It is noted that said length range is above the length range observed for tryptic peptides. As consequence, the present invention in this embodiment is distinguished from those prior art methods which make use of, for example, tryptic peptides or other peptides which are not amenable to cleavage by the proteolytic enzyme to be used for proteolytic digestion. Advantageously, and as stated above, subsequences in this length range give rise to a plurality of peptides upon proteolytic digestion, thereby enhancing accuracy of the quantitation.

The term “proteotypic” as used in this specific context refers to peptides which are frequently or always observed in the mass spectrum of a given polypeptide comprising said proteotypic peptide.

According to part (c) of this preferred embodiment, further features are provided which relate to the uniqueness of said subsequence (minimal sequence identity to other proteins, in particular to other proteins from the same proteome) or to easy handling and/or detection (exclusion of signal peptides and transmembrane segments).

In a further preferred embodiment, said known absolute amount of said tag polypeptide is determined by amino acid analysis. Preferred means and methods of amino acid analysis are described herein above.

In a fourth aspect, the present invention provides a fusion polypeptide for the quantification of a target polypeptide by mass spectroscopy, wherein: said fusion polypeptide consists of 35 to 455 amino acid residues and comprises (i) a target region, which is a fragment of the target polypeptide, and (ii) a tag region, which is not a fragment of the target polypeptide, said target region consists of 15 to 205 amino acid residues and comprises at least two signature regions; said tag region consists of 20 to 250 amino acid residues and comprises at least two signature regions; and each signature region has the structure Y-Z-X4-28-Y-Z, wherein all Y:s are selected from one of (i)-(iv), wherein (i) is R or K, (ii) is Y, F, W or L, (iii) is E and (iv) is D, and each X and each Z are independently any amino acid residue, provided that the Z:s are not P if the Y:s are selected from (i)-(iii); and each signature region comprises at least one amino acid residue comprising a heavy isotope.

This aspect relates to fusion polypeptides that may also be employed in the methods according to the invention. As throughout the specification, the target polypeptide may be any polypeptide, in particular a polypeptide naturally occurring in the proteome of any organism or cell in any state. The two regions comprised in the fusion polypeptide according to the fourth aspect of the invention are chosen such that each of them comprises at least two specific structural elements referred to as “signature regions”. Importantly, the N- and C-terminal amino acids of each signature region are selected such that they are recognized by a protease suitable for the mass spectrometry protocol described herein. The amino acids of (i)-(iv) are thus based on the selectivity of the following proteases: trypsin, which cleaves on the carboxyl side of arginine (R) and lysine (K) residues unless followed by proline (P); chymotrypsin, which cleaves on the carboxyl side of tyrosine (Y), phenylalanine (F), tryptophan (W) and leucine (L) residues unless followed by proline (P); Lys-C, which cleaves on the carboxyl side of lysine (K) residues unless followed by proline (P); Glu-C, which cleaves on the carboxyl side of glutamate (E) residues unless followed by proline (P); Arg-C, which cleaves on the carboxyl side of arginine (R) residues unless followed by proline (P); and Asp-N, which cleaves on the amino side of aspartate (D) residues. This design principle of the fusion polypeptides ensures that, upon proteolytic digestion, at least two mass-altered proteolytic products are obtained from the target and tag region, respectively. It is to be understood that the same Y residue may constitute the carboxylic end of a first signature region and the amino end of a second signature region.

The general term “mass-altered” is used herein as defined above. Preferably, it refers to a frequency of at least one isotope which deviates from the naturally occurring frequency/ies thereof, preferably to the exclusive occurrence of at least one heavy isotope, heavy isotopes preferably being selected from D, 13C and 15N.

In a preferred embodiment of the fusion polypeptide of the invention, said tag region or said tag polypeptide, respectively, corresponds to, i.e. comprises or consists of a solubility tag or a fragment thereof, said solubility tag being selected from Maltose-binding protein (MBP), Glutathione-S-transferase (GST), Thioredoxin (Trx), N-Utilization substance (NusA), Small ubiquitin-modifier (SUMO), a Solubility-enhancing tag (SET), a Disulfide forming protein C (DsbC), Seventeen kilodalton protein (Skp), Phage T7 protein kinase (T7PK), Protein G B1 domain (GB1), Protein A IgG ZZ repeat domain (ZZ) and Albumin Binding Protein (ABP). The structures of these solubility tags are known in the art and readily available to the skilled person. It follows from the above definition that the solubility tag (or fragment thereof) is mass-altered when constituting the tag region of the fusion polypeptide of the fourth aspect.

Preferably, said fragment is chosen such that the solubility conferring properties are retained or not significantly compromised. Whether or not this is the case can be determined by the skilled person without further ado, for example, by performing solubility assays for fusion constructs comprising a test polypeptide on the one hand and the solubility tag at issue or a fragment thereof on the other hand. By comparing solubility of constructs comprising the entire solubility tag with constructs comprising a fragment thereof, it can be determined whether and to which extent the solubility conferring properties are retained by the fragment under consideration.

For reasons discussed above, the sequences of the at least two signature regions of the tag region are, according to one embodiment, distinct from any sequence derivable from the human proteome by means of proteolysis.

The fusion polypeptide of the fourth aspect may for example be used in a diagnosis of a medical condition in a subject comprising the ex vivo quantification of a target polypeptide in a sample from the subject. Whenever human samples are analyzed, it may be beneficial if the tag region is not a human polypeptide. Thus, in an embodiment of the fourth aspect, the amino acid sequence of the tag region is not an amino acid sequence of a human protein or a fragment thereof. As human proteins may have high homology to proteins of other eukaryotes, it may be particularly preferred if the tag region has the amino acid sequence of a prokaryotic (e.g. bacterial) protein or a fragment thereof.

As already noted above, a particularly preferred tag region or tag polypeptide has the sequence set forth in SEQ ID NO: 1.

According to further preferred embodiments, said tag region consists of 40 to 150 amino acids, and independently said target region consists of 20 to 150 amino acids, such as 25 to 100 amino acids. Moreover, it is preferred that the fusion polypeptide consists of 80 to 300, more preferably 100 to 200 amino acids.

According to further preferred embodiments, said target region, and independently said tag region, comprises at least 3 such as at least 4, 5, 6, 7 or 8 signature regions. These preferred embodiments provide for an increasing number of proteolytic products to be formed from each of said regions when said fusion polypeptide is brought into contact with a proteolytic enzyme, proteolytic enzymes being further detailed below.

According to a further preferred embodiment, each signature region independently comprises at least 2, such as at least 3 or 4 amino acid residues comprising a heavy isotope.

LysC and trypsin has been found to be particularly suitable proteolytic enzymes (see e.g. the examples below). According to a further preferred embodiment, said Y:s are thus selected from R and K.

As stated above, preferred heavy isotopes are to be selected from deuterium (D), 13C and 15N.

Normally, the amino acid residues comprising a heavy isotope of the fusion polypeptide comprises more than one heavy isotope. A higher number of incorporated heavy isotopes may be preferred as it provides a larger mass shift. In a further preferred embodiment, the at least one amino acid residue comprising a heavy isotope is selected from L-arginine-13C6, L-arginine-13C615N4, L-arginine-13C615N4D7, L-arginine-15N4D7, L-arginine-15N4, L-lysine-13C615N2, L-lysine-15N2, L-lysine-13C6, L-lysine-13C615N2D9, L-lysine-15N2D9, L-lysine-D4, L-methionine-13CD3, L-tyrosine-13C9, L-tyrosine-15N and L-tyrosine-13C915N. Such heavy isotope labeled amino acids are well known in the art and available from a variety of manufacturers. The use of one or more of these amino acids is preferred for any labeling schemes and patterns according to the present invention. In a preferred mode, all lysines and arginines are labeled so that tryptic peptides typically contain one labeled amino acid as trypsin specifically cleaves C-terminally to arginine and lysine.

According to a further preferred embodiment, the fusion polypeptide further comprises a purification tag.

Moreover, to allow for an efficient expression of the fusion polypeptide, it is preferred that the target region of the fusion polypeptide does not correspond to a transmembrane spanning region of the target polypeptide. Further, it is also preferred that the target region of the fusion polypeptide does not correspond to a signal peptide of the target polypeptide, since the signal peptides are often cleaved off in a mature version of the target polypeptide.

In a preferred embodiment of any of the methods according to the invention as described above, said fusion polypeptide(s) is/are as defined in accordance with the fourth aspect of the present invention as well as embodiments referring back thereto.

Preferred purification tags are to be selected from His tag, a FLAG tag, a SBP tag, a myc tag and a OneStrep tag.

For a user quantifying one or more target proteins or polypeptides in a sample according to the present disclosure, it may be convenient to obtain the fusion polypeptide(s) necessary for the quantification preloaded onto a solid phase suitable for the proteolytic digestion. Such solid phase may be a solid support, a column or a filter. Preferably, the amount of fusion polypeptides on said support in the column is predetermined. Thus, the step of spiking the sample with the fusion polypeptide(s) is not in the responsibility of the user, which also reduces the risk of human error in the procedure. In a fifth aspect, the present invention thus furthermore relates to a column in or onto which at least one fusion polypeptide according to the fourth aspect is arranged. Means of arranging are within the skills of the skilled person and include covalent attachment as well as non-covalent adsorption or absorption.

A proteolytic enzyme such as trypsin, chymotrypsin, Lys-C, Glu-C or Asp-N may also be arranged in or onto the column. When using such a column, the user does not have to add the proteolytic enzyme for the digestion, which may be convenient and further reduce the risk of human error. According to one embodiment, the fusion polypeptide(s) are separated from the proteolytic enzyme on the support/in the column so as to prevent any proteolytic digestion before the sample is added.

The present invention in a sixth aspect provides a kit comprising: (a) at least one fusion polypeptide according to the fourth aspect; and (b) (i) a second polypeptide comprising or consisting of the same amino acid sequence as the tag region as defined in accordance with the fourth aspect but being differently isotope labeled compared to said tag region and/or (ii) a proteolytic enzyme, such as trypsin, chymotrypsin, Lys-C, Glu-C or Asp-N. The combination of the products necessary for the quantification protocol described herein into a kit may provide for increased reproducibility and decreased risk of human error at the users side. The second polypeptide of the sixth aspect may for example be “unlabeled”. It may also be “middle weight” or “extra heavy weight”. Such embodiments are discussed above in connection with the method aspects.

In a preferred embodiment of the kit, the at least one fusion polypeptide is arranged in or onto a column according to the fifth aspect of the invention. In a further preferred embodiment of the kit, said second polypeptide is provided in a known absolute amount.

In a further aspect, the present invention relates to use of a quantitative standard as defined in the second aspect or of a fusion polypeptide according to the fourth aspect of the invention as a reference in a target polypeptide quantification. In a preferred embodiment of the use according to the invention, said quantification is effected by mass spectrometry.

Various further embodiments of the use aspect are described in connection with the other aspects above.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures show:

FIG. 1: Schematic workflow for accurate determination of PrEST concentrations. Heavy or light ABP is recombinantly expressed in an auxotrophic E. coli strain and purified using the C-terminal OneStrep tag. The heavy labeled ABP, whose concentration is measured separately by amino acid analysis, and the PrEST are mixed together and an in-solution digest is performed. Peptides are measured with a short LC MS/MS run on a benchtop mass spectrometer and the PrEST concentration is accurately determined by the SILAC ratio of the ABP peptides originating from the PrEST and the ABP.

FIG. 2: Accuracy of ABP quantification. (A) Density plot of the overall distribution of the 43 coefficients of variation (CVs) of the ABP peptides measured on a benchtop Exactive mass spectrometer. (B) Representative example proteins showing the ratios of the ABP peptides and their coefficients of variation (CVs).

FIG. 3: Peptide ratio along the PrESTs sequences. The PrEST master mix was spiked into lysate of a cancer cell line and measured against the endogenous protein. The peptide ratios were extracted to quantify the proteins. The variation of the peptide ratios along the sequence is depicted. Overlapping peptides are due to missed cleavages.

FIG. 4: Reproducibility of the absolute quantification procedure. Three independent quantification experiments for representative examples, in which the master mix preparation as well as the PrEST quantification were performed independently. The bars reflect the median of the peptide ratios for each protein.

FIG. 5: Protein copy numbers determined per HeLa cell. The dot plot shows the protein copy numbers per cell measured in three independent experiments. The error bars correspond to the CVs. Proteins with copy numbers ranging from 4 000 to 20 000 000 per cell were quantified (see also Table 2).

FIG. 6: Direct quantification of a single protein in HeLa cell lysate. (A) Principle of the ‘single-plex’ strategy for the direct quantification of a single protein. In the same experiment, SILAC peptide ratios mapping to the ABP quantification tag determine the amount of PrEST whereas SILAC ratios mapping to the protein specific region of the PrEST construct determine the level of the endogenous proteins. The experiment can be performed with SILAC heavy labeled cells, unlabeled PrEST construct and heavy labeled ABP tag (left side) or vice versa (right side). (B) Single-plex determination of absolute protein amount. In the workflow depicted here, an unlabeled PrEST construct as well as a heavy labeled ABP tag are both spiked into HeLa cell lysate before digestion. (C) Comparison of copy numbers obtained from the ‘master mix’ experiment with those from the single-plex experiments for three different proteins. Error bars are standard deviations of the mean from triplicate measurements.

FIG. 7: Absolute Quantification using heavy PrESTs. (A) Comparison of copy numbers obtained by quantifying light PrESTs against SILAC labeled heavy cell lysate (black symbols) versus quantifying heavy PrESTs against unlabeled cell lysate (red symbols). (B) Values shown in A but plotted as a scatter graph.

FIG. 8: Comparison of SILAC-PrEST based quantification and ELISA. Proto-oncogene c-Fos (A) and Stratifin (B) were quantified by ELISA to evaluate the SILAC-PrEST absolute quantification. Different ELISA compatible buffers and filtered vs. unfiltered cell lysates were compared.

FIG. 9: Absolute quantification of the Integrin beta 3, Talin 1 and Kindlin 3 in different mice. (a) the integrin and its co-activators grouped together. (b) the decreasing expression levels of Kindlin 3 in comparison to the wild-type mice.

EXAMPLES

The examples illustrate the invention:

Example 1 Materials and Methods

Protein Epitope Signature Tags—The short protein fragments, i.e. the subsequences of target polypeptides, were produced in high-throughput by the Human Protein Atlas where they are used as antigens for antibody production (18, 19). In brief, suitable Protein Epitope Signature Tags (PrESTs) representing unique regions of each target protein were designed using the human genome sequence as template (EnsEMBL). Unique PrESTs with a size between 50 to 150 amino acids and low homology to other human proteins were selected, including epitope- and domain-sized similarities to other proteins, signal peptides and transmembrane regions (18). The cloning, protein expression and purification were performed as previously described (19, 20). For optimal storage PrESTs were lyophilized and dissolved in 8M urea and stored at −20° C. until further use. To ascertain that the PrESTs had an endogenous counterpart in HeLa cells, we selected 50 proteins spread over the abundance range of a HeLa proteome that we had measured at a depth of about 4,000 proteins. Proteins were picked without regards to specific protein classes, cellular localizations or functions. Of these 50 proteins, 43 were readily available from the Protein Atlas pipeline in recombinantly expressed form. For multiplexing experiments these 43 PrESTs were mixed together—each at the appropriate concentration. This ‘master mix’ that was then spiked into cell lysates.

Cell culture—For SILAC labeling, HeLa cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% dialyzed fetal bovine serum (Gibco) and penicillin/streptomycin (Gibco). Heavy arginine (high purity Arg10, Cambridge Isotope Laboratories) and heavy lysine (high purity Lys8, Cambridge Isotope Laboratory) were added to a final concentration of 33 μg/ml or 76 μg/ml, respectively. After six passages cells were fully labeled as assessed by mass spectrometry. Cells were counted using a Countess cell counter (Invitrogen) and aliquots of 106 cells were snap frozen and stored at −80° C.

Protein expression and purification of ABP (Albumin Binding Protein)—The expression vector pAff8c (Human Protein Atlas) was modified via SLIC cloning (21) inserting a OneStrep affinity tag to the C-terminus of the Albumin Binding Protein (ABP). To express heavy labeled ABP in E. coli, an expression strain auxotrophic for arginine and lysine was used (33). Cultures were grown in PA5052 minimal autoinduction media as previously described in (22) but with the addition of 18 normal (‘light’) amino acids and heavy arginine and lysine. Cultures were grown overnight and harvested at an OD600 of about 5.7. E. coli cells were lysed in 100 mM Tris, 150 mM NaCl and Protease Inhibitor (Roche) using a Bioruptor (Diagenode). Cell debris was removed by centrifugation and soluble ABP was purified using affinity chromatography on a StrepTap Hitrap column (GE Healthcare) coupled to an AKTA system. The purity of the protein was evaluated by mass spectrometry via an in solution digest followed by LC MS/MS. Abundances of ABP and contaminants were estimated by adding the signal for their most intense peptides. ABP was dialyzed in PBS, aliquoted, snap-frozen and stored at −80° C. The concentration of purified ABP was measured by amino acid analysis (Genaxxon BioScience GmbH).

Sample preparation—HeLa cells were lysed in 100 mM Tris, 4% SDS, 100 mM DTT, incubated for 5 min at 95° C. and disrupted using a Bioruptor. The lysate was cleared by centrifugation through SpinX filters (22 μm, Corning). The PrESTs were added at appropriate concentrations (see main text) to labeled HeLa cells and the samples were further processed by the FASP method (23). In brief, proteins were captured on a 30 kDa filter and SDS was exchanged with a urea containing buffer. Proteins were alkylated with iodoacetamide and trypsinzed (Promega). Further peptide separation was performed using pipette-based six fraction SAX as described (24).

The PrESTs and ABP were mixed and solubilized in denaturation buffer (6 M urea, 2 M thiourea in 10 mM HEPES, pH 8), reduced with DTT and subsequently alkylated with iodoacetamide. The protein mixture was digested with LysC (Wako) for 3 h, diluted with ammonium bicarbonate and further digested with trypsin overnight. The digestion was stopped by acidifying with TFA and desalted on C₁₋₈-Empore disc StageTips (25).

Liquid chromatography and mass spectrometry—Analysis of the light PrESTs spiked into HeLa cells was performed on a LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Easy nano-HPLC via a nanoelectrospray ion source (Proxeon Biosystems, now Thermo Fisher Scientific). The peptides were separated on a 15 cm fused silica emitter packed in-house with reversed phase material ReproSil-Pur 120 C18-AQ 3 μm resin (Dr. Maisch GmbH) and eluted with a 205 min gradient from 5-35% buffer B (80% acetonitrile, 0.5% acetic acid). The mass spectrometer was operated in a data dependent fashion to automatically measure MS and consecutive MS/MS. LTQ-Orbitrap full scan MS spectra (from 300 to 1650 m/z) were acquired with a resolution of 60,000 at m/z 400. The seven most abundant ions were sequentially isolated and fragmented in the linear ion trap using collision induced dissociation (CID) followed by analysis in the linear ion trap.

Analysis of the PrESTs spiked into HeLa cells was performed on an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) coupled to an Easy nano-HPLC via a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 20 cm column packed in-house using C18-AQ 1.8 μm resin (Dr. Maisch GmbH) and eluted with a 205-min gradient from 5-35% buffer B. The mass spectrometer was operated in a data dependent fashion to automatically measure MS and 10 consecutive MS/MS applying higher energy collision dissociation (HCD) (34). LTQ-Orbitrap full scan MS spectra (from 100 or 300 to 1650 m/z) were acquired with a resolution of 60,000 at m/z 400.

The PrEST-ABP peptides were analyzed online on the Exactive instrument with HCD option (Thermo Fisher Scientific) using the same nano-HPLC setup as described above. The peptides were eluted with a linear gradient with 5-30% buffer B over 40 min. The Exactive mass spectrometer identified peptides with All Ion Fragmentation (AIF) by performing alternating MS scans (300-1600 m/z) of the precursor ions and all ion fragmentation scans (100-1600 m/z) using stepped HCD fragmentation (26). Both scans were acquired at a resolution of 100 000 at m/z 200.

Data analysis—Acquired data were analyzed with MaxQuant (27) (version 1.1.1.36) using the human IPI database (v 3.68-87,083 entries). Common contaminants and the sequence of the ABP solubility tag were added to this database. For peptide identification we used Andromeda, a probabilistic search engine incorporated in to the MaxQuant framework (28). Carbamidomethylation of cysteine was included in the search as a fixed modification and methionine oxidation as well as N-terminal acetylation were included as variable modifications. We allowed two miscleavages and required a minimum of six amino acids per identified peptide. The initial mass tolerance for precursor ions or fragment ions was set to 6 ppm and fragment masses were allowed to deviate by up to 0.5 Th. For statistical evaluation of the data obtained, the posterior error probability and false discovery rate (FDR) were used. The FDR was determined by searching a reverse database and was set to 0.01 for peptide identification.

The AIF data was processed as described above except that up to 50 peaks were analyzed per 100 m/z with a tolerance of 15 ppm. The precursor ion mass was matched with the possible fragment ion candidates on the basis of the cosine correlation value of at least 0.6 (26).

Enzyme-linked Immunosorbent Assay—Absolute amounts measurements of proto-oncogene c-Fos and Stratifin (14-3-3σ) was carried out by ELISA. The kits were purchased from USCNK Life Science and performed according to the manufacturer's instructions. The HeLa cells were lysed in PBS, RIPA 1 (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40) or RIPA2 (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40; 0.1% SDS) with protease inhibitors. The cells were disrupted by 3 freeze-thaw cycles and sonication using the Biorupter. For the ELISA the samples were diluted 1:10. Fluorescence activity was measured by a microplate reader (Tecan) and converted to actual concentration by a standard curve.

Example 2 Absolute Quantification of Proteins in HeLa Cells

Unlike relative quantification, absolute quantification may be effected as a two step process that firstly requires measurement of the absolute amount of the standard and secondly the relative amount of the standard compared to the analyte of interest. Determination and subsequent control of the level of standard is by no means trivial and can easily be the step that limits the overall accuracy of the approach. Below, we first describe a generic method to determine the absolute amount of each PrEST with high accu racy.

Then we construct a ‘master mix’ of different PrESTs and evaluate the ability of the SILAC-PrEST method to accurately quantify cellular proteins. We then apply the master mix to determine the copy numbers of 37 proteins in a cancer cell line. Finally, we describe an alternative workflow for the quantification of single proteins of interest, in which the two steps are combined into one LC MS/MS analysis.

Accurate measurement of PrEST concentrations—Each PrEST is already fused to the Albumin Binding Domain (ABP), a solubilization tag of 120 amino acids. In silico digest of ABP results in 40 tryptic peptides with a length between 6 and 30 amino acids (Suppl. Table 1). We recombinantly expressed a heavy SILAC labeled version of the ABP protein tag. When necessary, we used a dual affinity approach based on an N-terminal His-tag and a C-terminal OneStrep tag to generate highly purified protein fragment and to ensure that only full length ABP was obtained. The absolute concentration of ABP protein fragment was determined by amino acid analysis, which is the most accurate method for protein quantification, but which is only applicable to highly purified proteins in relatively large amounts. Heavy SILAC incorporation into ABP was 99% and its purity was about 97% as judged by mass spectrometry (see Experimental Procedures). Because these two factors operate in a compensating direction and because of the small size of the effect, the measured concentration of ABP was not adjusted for them.

SUPPLEMENTARY TABLE 1 all ABP peptides detected in the AIF runs. All in silico peptides of the solubility tag ABP as well as the identified peptides when determining of  the accurate concentration of the PrEST (see FIG. 1) for all three master mixes. Peptide sequence Length Mass Missed cleavage detected TVEGVK 6 631.354 0 x NLINNAK 7 785.440 0 x SIELAEAK 8 859.465 0 x YGVSDYHK 8 967.440 0 x YGVSDYYK 8 993.444 0 x VLANRELDK 9 1056.593 1 x SQTPAEDTVK 10 1074.519 0 x DLQAQVVESAK 11 1186.619 0 x GSHMASLAEAK 11 1100.528 0 DLQAQVVESAKK 12 1314.714 1 x ELDKYGVSDYHK 12 1452.689 1 x ELDKYGVSDYYK 12 1478.693 1 x ISEATDGLSDFLK 13 1394.693 0 x NLINNAKTVEGVK 13 1398.783 1 SIELAEAKVLANR 13 1412.799 1 DLQAQVVESAKKAR 14 1541.853 2 ARISEATDGLSDFLK 15 1621.831 1 x YGVSDYHKNLINNAK 15 1734.869 1 YGVSDYYKNLINNAK 15 1760.873 1 GSHMASLAEAKVLANR 16 1653.862 1 KARISEATDGLSDFLK 16 1749.926 2 x MGSSHHHHHHSSGLVPR 17 1898.882 0 SIELAEAKVLANRELDK 17 1898.047 2 x TVEGVKDLQAQVVESAK 17 1799.963 1 x VLANRELDKYGVSDYHK 17 2006.022 2 x VLANRELDKYGVSDYYK 17 2032.027 2 x SQTPAEDTVKSIELAEAK 18 1915.974 1 x TVEGVKDLQAQVVESAKK 18 1928.058 2 x ELDKYGVSDYHKNLINNAK 19 2220.118 2 ELDKYGVSDYYKNLINNAK 19 2246.122 2 GGGSGGGSGGSAWSHPQFEK 20 1845.803 0 GSHMASLAEAKVLANRELDK 20 2139.111 2 YGVSDYHKNLINNAKTVEGVK 21 2348.213 2 YGVSDYYKNLINNAKTVEGVK 21 2374.217 2 ISEATDGLSDFLKSQTPAEDTVK 23 2451.202 1 x SQTPAEDTVKSIELAEAKVLANR 23 2469.308 2 NLINNAKTVEGVKDLQAQVVESAK 24 2567.392 2 ARISEATDGLSDFLKSQTPAEDTVK 25 2678.34 2 x ALIDEILAALPGTFAHYGSAWSHPQFEK 28 3068.54 0 MGSSHHHHHHSSGLVPRGSHMASLAEAK 28 2981.4 1

LC MS/MS of ABP indeed revealed many readily detectable tryptic peptides (see below). Each of the 43 PrESTs from the Protein Atlas Project was separately mixed with a known amount of labeled ABP as schematically outlined in FIG. 1 to allow for a SILAC LC-MS/MS experiment. As this experiment requires a separate LC MS/MS run for each PrEST it was likely to be rate limiting for the overall project.

We therefore decided to perform this analysis on an economical and robust benchtop Orbitrap instrument rather than on a Velos instrument. The Exactive instrument cannot isolate peptide precursors, therefore we identified the peptides by All Ion Fragmentation (AIF) (26) in 1 h runs. Typically, at least eight labeled ABP peptides could be quantified against the corresponding ABP peptides from the PrESTs, leading to a median coefficient of variation (CV) of 7% for PrEST quantification (FIG. 2A).

To overcome the step of measuring the PrESTs concentration, which limits overall throughput, the heavy PrESTs were measured by static nanoelectrospray on an automated chip-based system (TriVersa Nanomate). This enabled higher throughput measurements of these simple mixtures of ABP peptides using low sample consumption. The peptide ratio showed a median coefficient of variation 5.5%, an improvement over the Exactive based measurement of 7%.

Importantly, a particular PrEST quantification can be repeated at this stage until a desired accuracy is achieved. Here, this was not done, since the accuracy of PrEST quantification was estimated to be higher than that of the other steps in the workflow. A few typical examples of results from the PrEST quantification are shown in FIG. 2B. Note that the quantification accuracy does not depend on the cellular abundance or any other attributes of the target protein, since the same amounts of PrESTs is used in each PrEST quantification experiment. Importantly, quantification accuracy in our workflow also does not depend on the purity of the PrEST because our method specifically measures the concentration of PrEST and not of total protein.

PrEST master mix and endogenous protein quantification—Having quantified the PrEST amounts we proceeded to measuring protein expression levels in a human cancer cell line. For convenience we used unlabeled PrESTs and quantified against heavy SILAC labeled HeLa cells. Since digested total cell lysates consist of hundreds of thousands of tryptic peptides, the addition of a single or even a large number of PrEST does not change the overall complexity of the mixture. On the basis of the quantitative amounts established above, we here mixed 43 PrESTs together. In initial experiments we used equimolar mixtures of PrESTs, which were spiked into HeLa lysate in different amounts. The measured SILAC ratios established appropriate levels of each PrEST in the master mix, such that the SILAC ratios were within the most accurately quantifiable range, i.e. relatively close to one to one.

The master mix with appropriate levels of all the 43 PrESTs was spiked into the lysate of SILAC labeled cells. The mixture was digested according to the FASP protocol followed by SAX fractionation and resulting in six fractions that were separately measured with 4 h gradients on an LTQ Orbitrap mass spectrometer. We were able to quantify 37 of the 43 proteins targeted by our PrEST master mix.

Proteins were generally quantified with several PrEST derived peptides (average 3.7 and median 3), leading to an overall median CV of 18% (Supplementary Table 2). The results for these 37 protein targets are shown in FIG. 3 and the complete identification and quantification information is described in Supplementary Table 2. As an example, the adhesion protein IQGAP1 was quantified with five peptides, which each gave nearly identical quantification results (CV 10.6%). Six of the seven quantified tryptic peptides of ATP5B (mitochondrial ATP synthase subunit beta), had very close SILAC ratios, however, one peptide had a ratio that differs by 38% from the median. This peptide is clearly an outlier and its deviating value contributes substantially to the CV value, raising it from 8.2% to 27.2%. Note however, that we base protein quantification on the median of the peptide values; therefore the outlier peptide hardly contributes to the measured protein expression value and the CV value therefore underestimates the accuracy actually obtained in this experiment. For the same reason modifications of the endogenous proteins in the region covered by the PrEST could cause outlier peptide ratios, which would contribute little to the measured protein ratio.

SUPPLEMENTARY TABLE 2 All identification and quantification information used to quantify proteins. Ratio H/L Ratio H/L Ratio H/L Mastermix Mastermix Mastermix Protein Names Gene Name Sequence (1) CV(%) (2) CV(%) (3) CV(%) Cytosolic acyl coenzyme ACOT7 ADLPPCGACITGR NaN NaN — NaN Cytosolic acyl coenzyme ACOT7 GCCAPVQVVGPR NaN NaN NaN Cytosolic acyl coenzyme ACOT7 IMRPDDANVAGNVH 0.27622 0.25644 5.2343 GGTILK Cytosolic acyl coenzyme ACOT7 LVAGQGCVGPR NaN NaN NaN Cytosolic acyl coenzyme ACOT7 MIEEAGAIISTR NaN NaN NaN AFG3-like protein 2 AFG3L2 EQYLYTK 0.77684  6.27 0.82239   4.61 1.4433  5.83 AFG3-like protein 2 AFG3L2 HLSDSINQK 0.68028 0.78043 1.3793 AFG3-like protein 2 AFG3L2 LASLTPGFSGADVA 0.80176 0.82107 NaN NVCNEAALIA AFG3-like protein 2 AFG3L2 MCMTLGGR 0.72983 0.87038 1.5475 AFG3-like protein 2 AFG3L2 VSEEIFFGR 0.76345 0.87166 NaN ATPase family AAA dom ATAD2 DNFNFLHLNR 0.12868 — 0.12046 — 0.87365 — ATP synthase subunit b

ATP5B IMNVIGEPIDER 0.82979 23.09 0.64229 14.83 1.0596  9.57 ATP synthase subunit b

ATP5B IPVGPETLGR 0.85299 0.84263 1.1617 ATP synthase subunit b

ATP5B LVLEVAQHLGESTV 0.71767 0.73297 0.90287 R ATP synthase subunit b

ATP5B TIAMDGTEGLVR 0.4194 0.76715 1.1006 ATP synthase subunit b

ATP5B VLDSGAPIK 0.76219 0.67515 0.99543 ATP synthase subunit b

ATP5B VLDSGAPIKIPVGP 0.89528 0.95159 1.1652 ETLGR Zinc finger protein 828 C13orf8 ALFPEPR NaN 65.67 NaN 43.02 NaN 65.84 Zinc finger protein 828 C13orf8 AVELGDELQIDAID NaN NaN NaN DQK Zinc finger protein 828 C13orf8 CDILVQEELLASPK NaN NaN NaN Zinc finger protein 828 C13orf8 DNQESSDAELSSSE 0.081796 0.099583 0.34653 YIK Zinc finger protein 828 C13orf8 HALFPELPK NaN NaN NaN Zinc finger protein 828 C13orf8 KDNQESSDAELSSS NaN NaN NaN EYIK Zinc finger protein 828 C13orf8 LLEDTLFPSSK 0.22363 0.18665 0.9503 SRA stem-loop-interacti C14orf156 CILPFDK 3.5457 74.19 2.9157 76.61 1.466 40.99 SRA stem-loop-interacti C14orf156 EHFAQFGHVR NaN 2.9391 1.5112 SRA stem-loop-interacti C14orf156 GLGWVQFSSEEGLR 0.35422 0.33818 0.2253 SRA stem-loop-interacti C14orf156 IPWTAASSQLK NaN NaN 1.6141 SRA stem-loop-interacti C14orf156 NALQQENHIIDGVK 3.5138 3.226 1.5357 SRA stem-loop-interacti C14orf156 SINQPVAFVR NaN 0.29812 1.6403 μl μl pmol PrESTs PrESTs Exactive Exactive Exactive pmol pmol pmol pmol pmol Protein (1 + 2) (3) pmol/μl 1 pmol/μl 2 pmol/μl 3 PrEST 1 PrEST 2 PrEST 3 Protein 1 Protein 2 3 0.011 0.0017 275.36565 273.155 94.187082 3.029022 3.004705 0.160118 #VALUE! #VALUE! #VALUE! 0.011 0.0017 275.36565 273.155 94.187082 3.029022 3.004705 0.160118 #VALUE! #VALUE! #VALUE! 0.011 0.0017 275.36565 273.155 94.187082 3.029022 3.004705 0.160118 0.836676 0.770527 0.838106 0.011 0.0017 275.36565 273.155 94.187082 3.029022 3.004705 0.160118 #VALUE! #VALUE! #VALUE! 0.011 0.0017 275.36565 273.155 94.187082 3.029022 3.004705 0.160118 #VALUE! #VALUE! #VALUE! 0.006 0.0107 125.05216 129.519 17.806094 0.750313 0.777114 0.190525 0.582873 0.639091 0.274985 0.006 0.0107 125.05216 129.519 17.806094 0.750313 0.777114 0.190525 0.510423 0.606483 0.262791 0.006 0.0107 125.05216 129.519 17.806094 0.750313 0.777114 0.190525 0.601571 0.638065 #VALUE! 0.006 0.0107 125.05216 129.519 17.806094 0.750313 0.777114 0.190525 0.547601 0.676384 0.294838 0.006 0.0107 125.05216 129.519 17.806094 0.750313 0.777114 0.190525 0.572826 0.677379 #VALUE! 0.005 0.0025 179.90794 184.773 70.124349 0.89954 0.923865 0.175311 0.115753 0.111289 0.15316 0.049 0.0855 239.56298 196.145 81.16038 11.73859 9.611105 6.939212 9.740561 6.173117 7.35279 0.049 0.0855 239.56298 196.145 81.16038 11.73859 9.611105 6.939212 10.0129 8.098605 8.061283 0.049 0.0855 239.56298 196.145 81.16038 11.73859 9.611105 6.939212 8.424431 7.044652 6.265207 0.049 0.0855 239.56298 196.145 81.16038 11.73859 9.611105 6.939212 4.923163 7.373159 7.637297 0.049 0.0855 239.56298 196.145 81.16038 11.73859 9.611105 6.939212 8.947033 6.488938 6.9075 0.049 0.0855 239.56298 196.145 81.16038 11.73859 9.611105 6.939212 10.50932 9.145831 8.08557 0.007 0.0048 112.51212 82.565 38.582311 0.787585 0.577955 0.185195 #VALUE! #VALUE! #VALUE! 0.007 0.0048 112.51212 82.565 38.582311 0.787585 0.577955 0.185195 #VALUE! #VALUE! #VALUE! 0.007 0.0048 112.51212 82.565 38.582311 0.787585 0.577955 0.185195 #VALUE! #VALUE! #VALUE! 0.007 0.0048 112.51212 82.565 38.582311 0.787585 0.577955 0.185195 0.064421 0.057554 0.064176 0.007 0.0048 112.51212 82.565 38.582311 0.787585 0.577955 0.185195 #VALUE! #VALUE! #VALUE! 0.007 0.0048 112.51212 82.565 38.582311 0.787585 0.577955 0.185195 #VALUE! #VALUE! #VALUE! 0.007 0.0048 112.51212 82.565 38.582311 0.787585 0.577955 0.185195 0.176128 0.107875 0.175991 0.005 0.0378 151.61694 90.9 40.283794 0.758085 0.4545 1.522727 2.687941 1.325186 2.232318 0.005 0.0378 151.61694 90.9 40.283794 0.758085 0.4545 1.522727 #VALUE! 1.335821 2.301146 0.005 0.0378 151.61694 90.9 40.283794 0.758085 0.4545 1.522727 0.268529 0.153703 0.34307 0.005 0.0378 151.61694 90.9 40.283794 0.758085 0.4545 1.522727 #VALUE! #VALUE! 2.457834 0.005 0.0378 151.61694 90.9 40.283794 0.758085 0.4545 1.522727 2.663758 1.46649 2.338452 0.005 0.0378 151.61694 90.9 40.283794 0.758085 0.4545 1.522727 #VALUE! 0.135496 2.49773 Ratio H/L Ratio H/L Ratio H/L Mastermix Mastermix Mastermix Protein Names Gene Name Sequence (1) CV(%) (2) CV(%) (3) CV(%) Uncharacterized protein C1orf65 ILVELADEK NaN — NaN — NaN — Hepatocellular carcino

C9orf78 GDSESEEDEQDSEE NaN — NaN — NaN — VR Hepatocellular carcino

C9orf78 RGDSESEDEQDSEE NaN NaN NaN VR Hepatocellular carcino

C9orf78 VQEETTLVDDPFQM 0.34795 0.27853 0.54925 K Carbonyl reductase [NA CBR3 AFENCSEDLQER 0.12453  3.00 0.10329  9.75 2.5724 — Carbonyl reductase [NA CBR3 FHSETLTEGDLVDL 0.12993 NaN NaN MK Carbonyl reductase [NA CBR3 TNFFATR NaN 0.091036 NaN Carbonyl reductase [NA CBR3 VVNISSLQCLR NaN 0.11067 NaN Coiled-coil domain-cont CCDC55 NQEKPSNSESSLGA NaN — NaN — NaN — K T-complex protein 1 sub CCT2 HGINCFINR 0.63785 26.43 0.42168 30.21 1.3197  7.84 T-complex protein 1 sub CCT2 ILIANTGMDTDK 0.47498 0.37474 1.0965 T-complex protein 1 sub CCT2 ILIANTGMDTDKIK 0.26858 0.1831 NaN T-complex protein 1 sub CCT2 LALVTGGEIASTFD 0.5415 0.51676 1.2981 HPELVK T-complex protein 1 sub CCT2 LIEEVMIGEDK 0.34771 0.30789 1.2599 T-complex protein 1 sub CCT2 VAEIEHAEK 0.4707 0.45578 1.1598 T-complex protein 1 sub CCT2 VAEIEHAEKEK 0.51512 0.51219 1.3444 Charged multivesicular 

CHMP6 IAQQLER 0.12113 — 0.07044  9.14 NaN — Charged multivesicular 

CHMP6 YQEQLLDR NaN 0.080177 NaN COP9 signalosome com

COPS5 DHHYFK 0.9528  3.53 1.1093 19.40 1.7291 11.08 COP9 signalosome com

COPS5 ISALALLK 0.90507 0.82773 1.9194 COP9 signalosome com

COPS5 SGGNLEVMGLMLGK 0.96904 1.2254 2.157 COP9 signalosome com

COPS5 VDGETMIIMDSFAL NaN NaN NaN PVEGTETR Cytochrome b5 reducta CYB5R4 LLHDLNFSK NaN — NaN — NaN 34.50 Cytochrome b5 reducta CYB5R4 QGHISPALLSEFLK NaN NaN 0.077032 Cytochrome b5 reducta CYB5R4 TEDDIIWR 0.035422 0.034486 0.12675 Probable ATP-depender DDX20 GEEENMMMR 0.60662 — 0.59133 21.50 NaN — Probable ATP-depender DDX20 VLISTDLTSR NaN 0.43526 NaN μl μl pmol PrESTs PrESTs Exactive Exactive Exactive pmol pmol pmol pmol pmol Protein (1 + 2) (3) pmol/μl 1 pmol/μl 2 pmol/μl 3 PrEST 1 PrEST 2 PrEST 3 Protein 1 Protein 2 3 0.006 0.0026 221.44221 130.904 52.045542 1.328653 0.785424 0.135318 #VALUE! #VALUE! #VALUE! 0.006 0.0103 242.34528 179.229 78.04384 1.454072 1.075374 0.803852 #VALUE! #VALUE! #VALUE! 0.006 0.0103 242.34528 179.229 78.04384 1.454072 1.075374 0.803852 #VALUE! #VALUE! #VALUE! 0.006 0.0103 242.34528 179.229 78.04384 1.454072 1.075374 0.803852 0.505944 0.299524 0.441515 0.006 0.003 164.39734 155.761 70.484927 0.986384 0.934566 0.211455 0.122834 0.096531 0.543946 0.006 0.003 164.39734 155.761 70.484927 0.986384 0.934566 0.211455 0.128161 #VALUE! #VALUE! 0.006 0.003 164.39734 155.761 70.484927 0.986384 0.934566 0.211455 #VALUE! 0.085079 #VALUE! 0.006 0.003 164.39734 155.761 70.484927 0.986384 0.934566 0.211455 #VALUE! 0.103428 #VALUE! 0.015 0.0107 86.173017 86.118 38.073055 1.292595 1.29177 0.407382 #VALUE! #VALUE! #VALUE! 0.077 0.0951 337.27256 140.659 61.125623 25.96999 10.83074 5.813047 16.56496 4.567108 7.671478 0.077 0.0951 337.27256 140.659 61.125623 25.96999 10.83074 5.813047 12.33522 4.058713 6.374006 0.077 0.0951 337.27256 140.659 61.125623 25.96999 10.83074 5.813047 6.975019 1.983109 #VALUE! 0.077 0.0951 337.27256 140.659 61.125623 25.96999 10.83074 5.813047 14.06275 5.596895 7.545916 0.077 0.0951 337.27256 140.659 61.125623 25.96999 10.83074 5.813047 9.030024 3.334677 7.323858 0.077 0.0951 337.27256 140.659 61.125623 25.96999 10.83074 5.813047 12.22407 4.936436 6.741972 0.077 0.0951 337.27256 140.659 61.125623 25.96999 10.83074 5.813047 13.37766 5.547398 7.81506 0.011 0.0053 151.27677 86.581 38.633434 1.664044 0.952391 0.204757 0.201566 0.067086 #VALUE! 0.011 0.0053 151.27677 86.581 38.633434 1.664044 0.952391 0.204757 #VALUE! 0.07636 #VALUE! 0.004 0.0119 129.90234 97.939 31.757433 0.519609 0.391756 0.377913 0.495084 0.434575 0.65345 0.004 0.0119 129.90234 97.939 31.757433 0.519609 0.391756 0.377913 0.470283 0.324268 0.725367 0.004 0.0119 129.90234 97.939 31.757433 0.519609 0.391756 0.377913 0.503522 0.480058 0.815159 0.004 0.0119 129.90234 97.939 31.757433 0.519609 0.391756 0.377913 #VALUE! #VALUE! #VALUE! 0.006 0.0044 133.16874 85.926 35.243385 0.799012 0.515556 0.155071 #VALUE! #VALUE! #VALUE! 0.006 0.0044 133.16874 85.926 35.243385 0.799012 0.515556 0.155071 #VALUE! #VALUE! 0.011945 0.006 0.0044 133.16874 85.926 35.243385 0.799012 0.515556 0.155071 0.028303 0.017779 0.019655 0.005 0.0039 126.07369 113.423 42.562929 0.630368 0.567115 0.165995 0.382394 0.335352 #VALUE! 0.005 0.0039 126.07369 113.423 42.562929 0.630368 0.567115 0.165995 #VALUE! 0.246842 #VALUE! Ratio H/L Ratio H/L Ratio H/L Mastermix Mastermix Mastermix Protein Names Gene Name Sequence (1) CV(%) (2) CV(%) (3) CV(%) Enoyl-CoA hydratase, m ECHS1 EGMTAFVEK 0.19635 13.95 0.17962 14.70 1.3826  9.67 Enoyl-CoA hydratase, m ECHS1 ESVNAAFEMTLTE 0.14056 0.13711 1.0823 GSK Enoyl-CoA hydratase, m ECHS1 ICPVETLVEEAIQ 0.22122 0.19149 1.3474 CAEK Enoyl-CoA hydratase, m ECHS1 ISAQDAK NaN NaN NaN Enoyl-CoA hydratase, m ECHS1 IVVAMAK 0.15532 0.16412 NaN Enoyl-CoA hydratase, m ECHS1 KEGMTAFVEK NaN NaN NaN Enoyl-CoA hydratase, m ECHS1 KLFYSTFATDDR NaN 0.13372 NaN Enoyl-CoA hydratase, m ECHS1 LFYSTFATDDR 0.16792 0.15756 1.1803 Enoyl-CoA hydratase, m ECHS1 LFYSTFATDDRK 0.18416 0.20966 1.1801 Enoyl-CoA hydratase, m ECHS1 QAGLVSK 0.17537 0.16861 1.2314 Enoyl-CoA hydratase, m ECHS1 SLAMEMVLTGDR 0.17505 0.18633 1.0857 Eukaryotic translation i

EIF3E LGHVVMGNNAVSP 3.4941  5.39 1.4287 19.18 1.2643  7.57 YQQVIEK Eukaryotic translation i

EIF3E LNMTPEEAER NaN 1.877 1.431 Eukaryotic translation i

EIF3E SQMLAMNIEK 3.2375 NaN NaN Eukaryotic translation i

EIF3E WIVNLIR NaN NaN 1.253 Endoplasmic reticulum I ERLIN2 ADAECYTAMK 0.42437  8.66 0.39751 10.66 1.6231 18.18 Endoplasmic reticulum I ERLIN2 DIPNMFMDSAGSV 0.37141 0.35448 1.474 SK Endoplasmic reticulum I ERLIN2 LSFGLEDEPLETA 0.34781 0.35101 0.99961 TK Endoplasmic reticulum I ERLIN2 LTPEYLQLMK 0.43322 0.44968 1.6324 Endoplasmic reticulum I ERLIN2 QFEGLADK 0.37105 0.41697 1.5783 Endoplasmic reticulum I ERLIN2 VAQVAEITYGQK 0.40674 NaN NaN Fatty acid synthase FASN AALQEELQLCK 0.87952 13.38 0.79607 12.46 1.0964 15.17 Fatty acid synthase FASN DPSQQELPR 0.80543 NaN NaN Fatty acid synthase FASN FCFTPHTEEGCLS 0.79217 0.74449 1.0216 ER Fatty acid synthase FASN GLVQALQTK 0.91099 0.69768 1.262 Fatty acid synthase FASN LLSAACR 1.0665 NaN NaN Fatty acid synthase FASN MVVPGLDGAQIPR 0.77952 0.60727 1.1329 Fatty acid synthase FASN QQEQQVPILEK 0.73946 0.69517 0.8985 Fatty acid synthase FASN RQQEQQVPILEK 0.69837 0.67562 0.87805 Fatty acid synthase FASN VTQQGLK 0.83488 0.88813 1.1698 Fatty acid synthase FASN VTVAGGVHISGLH 0.71007 0.64471 0.82048 TESAPR μl μl pmol PrESTs PrESTs Exactive Exactive Exactive pmol pmol pmol pmol pmol Protein (1 + 2) (3) pmol/μl 1 pmol/μl 2 pmol/μl 3 PrEST 1 PrEST 2 PrEST 3 Protein 1 Protein 2 3 0.121 0.0495 232.98455 140.682 59.879442 28.19113 17.02252 2.964032 5.535328 3.057585 4.098071 0.121 0.0495 232.98455 140.682 59.879442 28.19113 17.02252 2.964032 3.962545 2.333958 3.207972 0.121 0.0495 232.98455 140.682 59.879442 28.19113 17.02252 2.964032 6.236442 3.259643 3.993737 0.121 0.0495 232.98455 140.682 59.879442 28.19113 17.02252 2.964032 #VALUE! #VALUE! #VALUE! 0.121 0.0495 232.98455 140.682 59.879442 28.19113 17.02252 2.964032 4.378646 2.793736 #VALUE! 0.121 0.0495 232.98455 140.682 59.879442 28.19113 17.02252 2.964032 #VALUE! #VALUE! #VALUE! 0.121 0.0495 232.98455 140.682 59.879442 28.19113 17.02252 2.964032 #VALUE! 2.276252 #VALUE! 0.121 0.0495 232.98455 140.682 59.879442 28.19113 17.02252 2.964032 4.733855 2.682069 3.498447 0.121 0.0495 232.98455 140.682 59.879442 28.19113 17.02252 2.964032 5.191679 3.568942 3.497855 0.121 0.0495 232.98455 140.682 59.879442 28.19113 17.02252 2.964032 4.943879 2.870167 3.649909 0.121 0.0495 232.98455 140.682 59.879442 28.19113 17.02252 2.964032 4.934857 3.171807 3.21805 0.004 0.0325 130.83738 149.56 50.714554 0.52335 0.59824 1.648223 1.828636 0.854705 2.083848 0.004 0.0325 130.83738 149.56 50.714554 0.52335 0.59824 1.648223 #VALUE! 1.122896 2.358607 0.004 0.0325 130.83738 149.56 50.714554 0.52335 0.59824 1.648223 1.694344 #VALUE! #VALUE! 0.004 0.0325 130.83738 149.56 50.714554 0.52335 0.59824 1.648223 #VALUE! #VALUE! 2.065223 0.005 0.0032 160.39251 113.243 49.792301 0.801963 0.566215 0.159335 0.340329 0.225076 0.258617 0.005 0.0032 160.39251 113.243 49.792301 0.801963 0.566215 0.159335 0.297857 0.200712 0.23486 0.005 0.0032 160.39251 113.243 49.792301 0.801963 0.566215 0.159335 0.278931 0.198747 0.159273 0.005 0.0032 160.39251 113.243 49.792301 0.801963 0.566215 0.159335 0.347426 0.254616 0.260099 0.005 0.0032 160.39251 113.243 49.792301 0.801963 0.566215 0.159335 0.297568 0.236095 0.251479 0.005 0.0032 160.39251 113.243 49.792301 0.801963 0.566215 0.159335 0.232619 #VALUE! #VALUE! 0.066 0.1232 127.7533 101.472 43.487725 8.431718 6.697152 5.357688 7.415865 5.331402 5.874169 0.066 0.1232 127.7533 101.472 43.487725 8.431718 6.697152 5.357688 6.791159 #VALUE! #VALUE! 0.066 0.1232 127.7533 101.472 43.487725 8.431718 6.697152 5.357688 6.679354 4.985963 5.473414 0.066 0.1232 127.7533 101.472 43.487725 8.431718 6.697152 5.357688 7.681211 4.672469 6.761402 0.066 0.1232 127.7533 101.472 43.487725 8.431718 6.697152 5.357688 8.992427 #VALUE! #VALUE! 0.066 0.1232 127.7533 101.472 43.487725 8.431718 6.697152 5.357688 6.572693 4.066979 6.069724 0.066 0.1232 127.7533 101.472 43.487725 8.431718 6.697152 5.357688 6.234918 4.655659 4.813882 0.066 0.1232 127.7533 101.472 43.487725 8.431718 6.697152 5.357688 5.888459 4.52473 4.704318 0.066 0.1232 127.7533 101.472 43.487725 8.431718 6.697152 5.357688 7.039473 5.947942 6.267423 0.066 0.1232 127.7533 101.472 43.487725 8.431718 6.697152 5.357688 5.98711 4.317721 4.395876 Ratio H/L Ratio H/L Ratio H/L Mastermix Mastermix Mastermix Protein Names Gene Name Sequence (1) CV(%) (2) CV(%) (3) CV(%) Flap endonuclease 1 FEN1 AVDLIQK 0.72265 19.67 0.74027 21.43 1.2719 10.53 Flap endonuclease 1 FEN1 EQHQLFLEPEVLD 0.85331 0.83635 1.3723 PESVELK Flap endonuclease 1 FEN1 HLTASEAK 0.69703 0.63769 1.0235 Flap endonuclease 1 FEN1 LDPNKYPVPENWL 0.73377 0.68 1.2048 HK Flap endonuclease 1 FEN1 LPIQEFHLSR 0.65281 0.4747 1.0466 Flap endonuclease 1 FEN1 SIEEIVR 1.1354 NaN NaN Flap endonuclease 1 FEN1 VYAAATEDMDCLT 0.73515 0.45581 1.1345 FGSPVLMR Flap endonuclease 1 FEN1 YPVPENWLHK 0.72303 0.70528 1.1416 Cellular oncogene fos; G FOS ELTDTLQAETDQL NaN — NaN 26.11 NaN  4.68 EDEK Cellular oncogene fos; G FOS GKVEQLSPEEEEK NaN 0.0090707 0.050604 Cellular oncogene fos; G FOS SALQTEIANLLK 0.015533 0.013178 0.054066 Cellular oncogene fos; G FOS VEQLSPEEEEK NaN NaN NaN Heat shock 70 kDa prot

HSPA4 EDQYDHLDAADMT 0.2685 78.77 0.20351  7.31 0.79843 13.41 K Heat shock 70 kDa prot

HSPA4 LNLQNK 0.25909 0.21666 0.94161 Heat shock 70 kDa prot

HSPA4 NKEDQYDHLDAAD 0.21899 0.21981 0.84382 MTK Heat shock 70 kDa prot

HSPA4 QIQQYMK 0.95485 NaN NaN Heat shock 70 kDa prot

HSPA4 QSLTMDPVVK 0.24663 0.22089 0.89363 Heat shock 70 kDa prot

HSPA4 STNEAMEWMNNK 0.25098 0.24797 1.1176 Ras GTPase-activiting-l

QGAP1 ILAIGLINEALDE 0.26959  9.98 0.27248  5.48 1.4819  6.12 GDAQK Ras GTPase-activiting-l

QGAP1 LEGVLAEVAQHYQ 0.27809 0.273 1.4224 DTLIR Ras GTPase-activiting-l

QGAP1 QLSSSVTGLTNIE 0.27918 0.23809 1.4471 EENCQR Ras GTPase-activiting-l

QGAP1 TLQALQIPAAK 0.218 0.25878 1.5877 Ras GTPase-activiting-l

QGAP1 YLDELMK 0.27889 0.26493 1.3441 Mitogen-activated prot

MAP2K1IP1 ELAPLFEELR 0.32694 — 0.056059 — 1.6953 27.91 Mitogen-activated prot

MAP2K1IP1 KLPSVEGLHAIVV NaN NaN 1.1365 SDR Mitogen-activated prot

MAP2K1IP1 VANDNAPEHALRP NaN NaN NaN GFLSTFALATI Mixed lineage kinase do MLKL APVAIK 0.39067 — NaN — 0.89538 39S ribosomal protein L MRPL50 AYTPPEDLQSR 0.40095  2.84 NaN  5.03 1.0384 42.41 39S ribosomal protein L MRPL50 EKEPVVVETVEEK 0.41737 0.67673 2.6456 39S ribosomal protein L MRPL50 LESYVK NaN 0.63021 2.3002 μl μl pmol PrESTs PrESTs Exactive Exactive Exactive pmol pmol pmol pmol pmol Protein (1 + 2) (3) pmol/μl 1 pmol/μl 2 pmol/μl 3 PrEST 1 PrEST 2 PrEST 3 Protein 1 Protein 2 3 0.035 0.061 155.06238 141.409 37.29001 5.427183 4.494315 2.274691 3.921954 3.663829 2.893179 0.035 0.061 155.06238 141.409 37.29001 5.427183 4.494315 2.274691 4.63107 4.13936 3.121558 0.035 0.061 155.06238 141.409 37.29001 5.427183 4.494315 2.274691 3.78291 3.156129 2.328146 0.035 0.061 155.06238 141.409 37.29001 5.427183 4.494315 2.274691 3.982304 3.365534 2.740547 0.035 0.061 155.06238 141.409 37.29001 5.427183 4.494315 2.274691 3.54292 2.34944 2.380691 0.035 0.061 155.06238 141.409 37.29001 5.427183 4.494315 2.274691 6.162024 #VALUE! #VALUE! 0.035 0.061 155.06238 141.409 37.29001 5.427183 4.494315 2.274691 3.989794 2.255947 2.580637 0.035 0.061 155.06238 141.409 37.29001 5.427183 4.494315 2.274691 3.924016 3.490653 2.596787 0.007 0.0033 151.55943 141.201 52.196632 1.060916 0.988407 0.172249 #VALUE! #VALUE! #VALUE! 0.007 0.0033 151.55943 141.201 52.196632 1.060916 0.988407 0.172249 #VALUE! 0.008966 0.008716 0.007 0.0033 151.55943 141.201 52.196632 1.060916 0.988407 0.172249 0.016479 0.013025 0.009313 0.007 0.0033 151.55943 141.201 52.196632 1.060916 0.988407 0.172249 #VALUE! #VALUE! #VALUE! 0.073 0.0447 190.231 154.209 68.382353 13.88686 11.25726 3.056691 3.728623 2.290964 2.440554 0.073 0.0447 190.231 154.209 68.382353 13.88686 11.25726 3.056691 3.597947 2.438997 2.878211 0.073 0.0447 190.231 154.209 68.382353 13.88686 11.25726 3.056691 3.041084 2.474458 2.579297 0.073 0.0447 190.231 154.209 68.382353 13.88686 11.25726 3.056691 13.25987 #VALUE! #VALUE! 0.073 0.0447 190.231 154.209 68.382353 13.88686 11.25726 3.056691 3.424917 2.486615 2.731551 0.073 0.0447 190.231 154.209 68.382353 13.88686 11.25726 3.056691 3.485325 2.791462 3.416158 0.109 0.054 98.606097 72.632 27.555556 10.74806 7.916888 1.488 2.897571 2.157194 2.205067 0.109 0.054 98.606097 72.632 27.555556 10.74806 7.916888 1.488 2.988929 2.16131 2.116531 0.109 0.054 98.606097 72.632 27.555556 10.74806 7.916888 1.488 3.000645 1.884932 2.153285 0.109 0.054 98.606097 72.632 27.555556 10.74806 7.916888 1.488 2.343078 2.048732 2.362498 0.109 0.054 98.606097 72.632 27.555556 10.74806 7.916888 1.488 2.997528 2.097421 2.000021 0.006 0.0033 149.10541 128.996 49.884398 0.894632 0.773976 0.164619 0.292491 0.043388 0.279078 0.006 0.0033 149.10541 128.996 49.884398 0.894632 0.773976 0.164619 #VALUE! #VALUE! 0.187089 0.006 0.0033 149.10541 128.996 49.884398 0.894632 0.773976 0.164619 #VALUE! #VALUE! #VALUE! 0.005 0.0048 105.25905 95.876 38.828103 0.526295 0.47938 0.186375 0.205608 #VALUE! 0.166876 0.006 0.0042 121.96997 107.299 33.817122 0.73182 0.643794 0.142032 0.293423 #VALUE! 0.147486 0.006 0.0042 121.96997 107.299 33.817122 0.73182 0.643794 0.142032 0.30544 0.435675 0.37576 0.006 0.0042 121.96997 107.299 33.817122 0.73182 0.643794 0.142032 #VALUE! 0.405725 0.326702 Ratio H/L Ratio H/L Ratio H/L Mastermix Mastermix Mastermix Protein Names Gene Name Sequence (1) CV(%) (2) CV(%) (3) CV(%) 28S ribosomal protein S MRPS23 ALLAEGVILR 0.33211 7.87 0.30866 51.08 2.8825  3.38 28S ribosomal protien S MRPS23 LGETDEEK 0.31151 0.33385 2.7777 28S ribosomal protein S MRPS23 TQHGGSHVSR 0.36147 0.061638 2.6945 28S ribosomal protein S MRPS23 YTELQK 0.30384 0.3357 NaN 28S ribosomal protein S MRPS23 AGGFASALER 0.79424 28.44 0.59711 14.13 NaN 11.34 28S ribosomal protein S MRPS23 HSELLQK 0.48648 0.62859 1.2025 28S ribosomal protein S MRPS23 NVESFASMLR 0.87278 0.77368 1.4122 Purine nucleoside phos

NP ACVMMQGR 0.65455 25.15 0.63682 21.02 0.89372  8.08 Purine nucleoside phos

NP DHINLPGFSGQNP 0.57033 0.47254 0.98592 LR Purine nucleoside phos

NP FEVGDIMLIR 0.56081 0.55235 0.98611 Purine nucleoside phos

NP FHMYEGYPLWK 0.58408 0.57218 0.85084 Purine nucleoside phos

NP HRPQVAIICGSGL NaN 0.50154 0.84573 GGLTDK Purine nucleoside phos

NP LTQAQIFDYGEIP 0.26064 0.27356 NaN NFPR Purine nucleoside phos

NP STVPGHAGR 0.54236 0.51975 0.79355 Purine nucleoside phos

NP VFHLLGVDTLVVT 0.68813 0.51622 0.88994 NAAGGLNPK Poly[ADP-ribose]poly

PARP4 ADLCQLIR 0.18498 49.36 0.18395  6.50 NaN Poly[ADP-ribose]poly

PARP4 AEGILLLVK NaN NaN NaN Poly[ADP-ribose]poly

PARP4 EVNLGLLAK 0.089268 0.16778 NaN Prefoldin subunit 1 PFDN1 EAEDNIR NaN  2.89 NaN 12.33 NaN  8.38 Prefoldin subunit 1 PFDN1 EAIHSQLLEK 0.17447 0.1967 1.8144 Prefoldin subunit 1 PFDN1 IKELEQK NaN NaN NaN Prefoldin subunit 1 PFDN1 LADIQIEQLNR 0.18176 0.16018 1.6113 Prefoldin subunit 1 PFDN1 MFILQSK NaN 0.20263 NaN Peptidyl-prolyl cis-trans PPIB DKPLKDVIIADCG 0.47962  7.94 0.41764  7.16 1.0739  7.51 K Peptidyl-prolyl cis-trans PPIB DTNGSQFFITTVK 0.50189 0.47562 1.2206 Peptidyl-prolyl cis-trans PPIB DVIIADCGK 0.5755 0.51014 1.3142 Peptidyl-prolyl cis-trans PPIB IEVEKPFAIAK 0.46154 0.453 1.1893 Peptidyl-prolyl cis-trans PPIB TAWLDGK 0.49743 0.49432 1.2376 Peptidyl-prolyl cis-trans PPIB VLEGMEVVR 0.48337 0.49474 1.3259 μl μl Exactive pmol PrESTs PrESTs Exactive pmol/ Exactive pmol pmol pmol pmol pmol Protein (1 + 2) (3) pmol/μl 1 μl 2 pmol/μl 3 PrEST 1 PrEST 2 PrEST 3 Protein 1 Protein 2 3 0.006 0.0024 198.09216 181.075 69.643134 1.188553 1.08645 0.167144 0.39473 0.335344 0.481791 0.006 0.0024 198.09216 181.075 69.643134 1.188553 1.08645 0.167144 0.370246 0.362711 0.464275 0.006 0.0024 198.09216 181.075 69.643134 1.188553 1.08645 0.167144 0.429626 0.066967 0.450368 0.006 0.0024 198.09216 181.075 69.643134 1.188553 1.08645 0.167144 0.36113 0.364721 #VALUE! 0.005 0.0081 191.91632 145.872 66.25803 0.959582 0.72936 0.53669 0.762138 0.435508 #VALUE! 0.005 0.0081 191.91632 145.872 66.25803 0.959582 0.72936 0.53669 0.466817 0.458468 0.64537 0.005 0.0081 191.91632 145.872 66.25803 0.959582 0.72936 0.53669 0.837504 0.564291 0.757914 0.055 0.0864 112.22478 79.837 33.590734 6.172363 4.391035 2.902239 4.04012 2.796299 2.593789 0.055 0.0864 112.22478 79.837 33.590734 6.172363 4.391035 2.902239 3.520284 2.07494 2.861376 0.055 0.0864 112.22478 79.837 33.590734 6.172363 4.391035 2.902239 3.461523 2.425388 2.861927 0.055 0.0864 112.22478 79.837 33.590734 6.172363 4.391035 2.902239 3.605154 2.512462 2.469341 0.055 0.0864 112.22478 79.837 33.590734 6.172363 4.391035 2.902239 #VALUE! 2.20228 2.454511 0.055 0.0864 112.22478 79.837 33.590734 6.172363 4.391035 2.902239 1.608765 1.201212 #VALUE! 0.055 0.0864 112.22478 79.837 33.590734 6.172363 4.391035 2.902239 3.347643 2.28224 2.303072 0.055 0.0864 112.22478 79.837 33.590734 6.172363 4.391035 2.902239 4.247388 2.26674 2.582819 0.005 0.0037 144.98538 124.938 55.100415 0.724927 0.62469 2.203872 0.134097 0.114912 #VALUE! 0.005 0.0037 144.98538 124.938 55.100415 0.724927 0.62469 2.203872 #VALUE! #VALUE! #VALUE! 0.005 0.0037 144.98538 124.938 55.100415 0.724927 0.62469 2.203872 0.064713 0.10481 #VALUE! 0.023 0.0071 193.04964 191.964 33.110306 4.440142 4.415172 0.235083 #VALUE! #VALUE! #VALUE! 0.023 0.0071 193.04964 191.964 33.110306 4.440142 4.415172 0.235083 0.774672 0.868464 0.426535 0.023 0.0071 193.04964 191.964 33.110306 4.440142 4.415172 0.235083 #VALUE! #VALUE! #VALUE! 0.023 0.0071 193.04964 191.964 33.110306 4.440142 4.415172 0.235083 0.80704 0.707222 0.37879 0.023 0.0071 193.04964 191.964 33.110306 4.440142 4.415172 0.235083 #VALUE! 0.894646 #VALUE! 0.531 0.6195 99.552439 58.765 22.904459 52.86234 31.20422 14.18931 25.35384 13.03213 15.2379 0.531 0.6195 99.552439 58.765 22.904459 52.86234 31.20422 14.18931 26.53108 14.84135 17.31947 0.531 0.6195 99.552439 58.765 22.904459 52.86234 31.20422 14.18931 30.42228 15.91852 18.64759 0.531 0.6195 99.552439 58.765 22.904459 52.86234 31.20422 14.18931 24.39809 14.13551 16.87535 0.531 0.6195 99.552439 58.765 22.904459 52.86234 31.20422 14.18931 26.29532 15.42487 17.56069 0.531 0.6195 99.552439 58.765 22.904459 52.86234 31.20422 14.18931 25.55207 15.43797 18.81361 Ratio H/L Ratio H/L Ratio H/L Mastermix Mastermix Mastermix Protein Names Gene Name Sequence (1) CV(%) (2) CV(%) (3) CV(%) Peroxiredoxin 6 PRDX6 DFTPVCTTELGR 0.70188 6.96 0.68855  7.92 0.98276  9.15 Peroxiredoxin 6 PRDX6 DINAYNCEEPTEK 0.75055 0.74788 1.1286 Peroxiredoxin 6 PRDX6 ELAILLGMLDPAEK 0.77082 0.61275 1.2449 Peroxiredoxin 6 PRDX6 ELAILLGMLDPAEK 0.62879 0.65579 1.2636 DEK Peroxiredoxin 6 PRDX6 FHDFLGDSWGILFS NaN 0.78925 NaN HPR Peroxiredoxin 6 PRDX6 GMPVTAR 0.71311 0.79113 1.0751 Peroxiredoxin 6 PRDX6 LAPEFAK 0.73135 0.65758 1.1849 Peroxiredoxin 6 PRDX6 LIALSIDSVEDHLA 0.72068 0.73169 1.1426 WSK Peroxiredoxin 6 PRDX6 LPFPIIDDR 0.74256 0.74049 1.1585 Peroxiredoxin 6 PRDX6 VVFVFGPDK 0.6815 0.74577 1.1822 Peroxiredoxin 6 PRDX6 VVFVFGPDKK 0.81434 0.72122 0.94586 26S protease regulatory PSMC3 AVCVEAGMIALR 0.89983 24.43 0.51761 10.16 NaN 26S protease regulatory PSMC3 GATELTHEDYMEGI NaN NaN NaN LEVQAK 26S protease regulatory PSMC3 IMQIHSR 0.58166 0.60039 NaN 26S protease regulatory PSMC3 MNVSPDVNYEELAR 0.62842 0.63254 1.0965 14-3-3 protein sigma SFN IIDSAR NaN  7.23 NaN  8.77 NaN 16.05 14-3-3 protein sigma SFN SAYQEAMDISK 0.41269 0.41814 1.1131 14-3-3 protein sigma SFN SNEEGSEEKGPEVR 0.3446 0.35575 0.75982 14-3-3 protein sigma SFN VETELQGVCDTVLG NaN 0.43773 NaN LLDSHLIK 14-3-3 protein sigma SFN VLSSIEQK 0.39497 0.35675 0.87046 14-3-3 protein sigma SFN YLAEVATGDDK 0.41281 0.38665 0.98489 14-3-3 protein sigma SFN YLAEVATGDDKK 0.404 0.41942 0.79403 FACT complex subunit S SSRP1 ADVIQATGDAICIF 0.85761  3.66 0.73149  6.06 1.1277  5.26 R FACT complex subunit S SSRP1 ELQCLTPR 0.90583 0.73695 1.2494 FACT complex subunit S SSRP1 IPYTTVLR 0.84574 0.79479 1.2167 FACT complex subunit S SSRP1 LFLLPHK NaN 0.68587 NaN THO complex subunit 1 THOC1 AVNNSNYGWR 0.43053 26.28 0.34141 24.03 NaN 43.19 THO complex subunit 1 THOC1 LWNLCPDNMEACK 0.55515 0.38786 2.8589 THO complex subunit 1 THOC1 SLPEYLENMVIK 0.50412 0.38951 2.9525 THO complex subunit 1 THOC1 TGEDEDEEDNDALL 0.28678 0.21856 1.1674 K Nucleoprotein TPR TPR ILLSQTTGVAIPLH 0.1447  7.15 0.16851 10.98 NaN 16.59 ASSLDDVSLAS Nucleoprotein TPR TPR ITELQLK 0.17454 0.16679 1.6804 μl μl Exactive pmol PrESTs PrESTs Exactive pmol/ Exactive pmol pmol pmol pmol pmol Protein (1 + 2) (3) pmol/μl 1 μl 2 pmol/μl 3 PrEST 1 PrEST 2 PrEST 3 Protein 1 Protein 2 3 0.202 0.3388 100.65311 94.21 37.403786 20.33193 19.03042 12.6724 14.27057 13.1034 12.45393 0.202 0.3388 100.65311 94.21 37.403786 20.33193 19.03042 12.6724 15.26013 14.23247 14.30207 0.202 0.3388 100.65311 94.21 37.403786 20.33193 19.03042 12.6724 15.67226 11.66089 15.77587 0.202 0.3388 100.65311 94.21 37.403786 20.33193 19.03042 12.6724 12.78451 12.47996 16.01285 0.202 0.3388 100.65311 94.21 37.403786 20.33193 19.03042 12.6724 #VALUE! 15.01976 #VALUE! 0.202 0.3388 100.65311 94.21 37.403786 20.33193 19.03042 12.6724 14.4989 15.05554 13.6241 0.202 0.3388 100.65311 94.21 37.403786 20.33193 19.03042 12.6724 14.86976 12.51402 15.01553 0.202 0.3388 100.65311 94.21 37.403786 20.33193 19.03042 12.6724 14.65281 13.92437 14.47949 0.202 0.3388 100.65311 94.21 37.403786 20.33193 19.03042 12.6724 15.09768 14.09184 14.68098 0.202 0.3388 100.65311 94.21 37.403786 20.33193 19.03042 12.6724 13.85621 14.19232 14.98131 0.202 0.3388 100.65311 94.21 37.403786 20.33193 19.03042 12.6724 16.5571 13.72512 11.98632 0.017 0.0294 167.07191 156.456 61.444877 2.840222 2.659752 1.806479 2.555717 1.376714 #VALUE! 0.017 0.0294 167.07191 156.456 61.444877 2.840222 2.659752 1.806479 #VALUE! #VALUE! #VALUE! 0.017 0.0294 167.07191 156.456 61.444877 2.840222 2.659752 1.806479 1.652044 1.596889 #VALUE! 0.017 0.0294 167.07191 156.456 61.444877 2.840222 2.659752 1.806479 1.784853 1.6824 1.980805 0.096 0.102 100.90731 80.163 30.014189 9.687101 7.695648 3.061447 #VALUE! #VALUE! #VALUE! 0.096 0.102 100.90731 80.163 30.014189 9.687101 7.695648 3.061447 3.99777 3.217858 3.407697 0.096 0.102 100.90731 80.163 30.014189 9.687101 7.695648 3.061447 3.338175 2.737727 2.326149 0.096 0.102 100.90731 80.163 30.014189 9.687101 7.695648 3.061447 #VALUE! 3.368616 #VALUE! 0.096 0.102 100.90731 80.163 30.014189 9.687101 7.695648 3.061447 3.826114 2.745422 2.664867 0.096 0.102 100.90731 80.163 30.014189 9.687101 7.695648 3.061447 3.998932 2.975522 3.015189 0.096 0.102 100.90731 80.163 30.014189 9.687101 7.695648 3.061447 3.913589 3.227709 2.430881 0.017 0.0343 128.21107 139.714 48.156415 2.179588 2.375138 1.651765 1.869237 1.73739 1.862695 0.017 0.0343 128.21107 139.714 48.156415 2.179588 2.375138 1.651765 1.974336 1.750358 2.063715 0.017 0.0343 128.21107 139.714 48.156415 2.179588 2.375138 1.651765 1.843365 1.887736 2.009703 0.017 0.0343 128.21107 139.714 48.156415 2.179588 2.375138 1.651765 #VALUE! 1.629036 #VALUE! 0.006 0.0036 133.17121 131.715 33.493744 0.799027 0.79029 0.120577 0.344005 0.269813 #VALUE! 0.006 0.0036 133.17121 131.715 33.493744 0.799027 0.79029 0.120577 0.44358 0.306522 0.344719 0.006 0.0036 133.17121 131.715 33.493744 0.799027 0.79029 0.120577 0.402806 0.307826 0.356005 0.006 0.0036 133.17121 131.715 33.493744 0.799027 0.79029 0.120577 0.229145 0.172726 0.140762 0.021 0.0086 186.99624 129.566 51.550628 3.926921 2.720886 0.443335 0.568225 0.458496 #VALUE! 0.021 0.0086 186.99624 129.566 51.550628 3.926921 2.720886 0.443335 0.685405 0.453817 0.744981 Ratio H/L Ratio H/L Ratio H/L Mastermix Mastermix Mastermix Protein Names Gene Name Sequence (1) CV(%) (2) CV(%) (3) CV(%) Nucleoprotein TPR TPR LESALTELEQLR 0.1666 0.15943 1.1901 Nucleoprotein TPR TPR LESALTELEQLRK 0.17493 0.17872 1.3357 Nucleoprotein TPR TPR NIEELQQQNQR 0.17718 0.18774 1.3128 Nucleoprotein TPR TPR QHQMQLVDSIVR 0.16848 0.21389 1.7312 Cytochrome b-c1 compl UQCRC1 ADLTEYLSTHYK 1.6066  5.68 1.5721  5.68 1.3068 12.57 Cytochrome b-c1 compl UQCRC1 DVVFNYLHATAFQG 1.524 1.4183 1.3969 TPLAQAVTC Cytochrome b-c1 compl UQCRC1 MVLAAAGGVEHQQL 1.707 1.5624 1.6586 LDLAQK Transitional endoplasmi VCP DHFEEAMR 0.20879 29.20 0.20611 21.24 1.4096 26.47 Transitional endoplasmi VCP ESIESEIR 0.075211 0.16428 NaN Transitional endoplasmi VCP GFGSFR 0.21737 0.22985 1.287 Transitional endoplasmi VCP KYEMFAQTLQQSR 0.12676 NaN 0.47634 Transitional endoplasmi VCP MTNGFSGADLTEIC 0.23492 0.2695 1.5438 QR Transitional endoplasmi VCP QTNPSAMEVEEDDP 0.14391 0.15975 1.292 VPEIR Transitional endoplasmi VCP RDHFEEAMR 0.20524 0.17819 1.459 Transitional endoplasmi VCP SVSDNDIR 0.19148 0.15796 1.2779 Transitional endoplasmi VCP YEMFAQTLQQSR 0.20698 0.24338 1.4679 Vimentin VIM DNLAEDIMR 0.813 15.32 0.79552 16.24 1.6492 33.78 Vimentin VIM EEAENTLQSFR 0.78127 0.81105 1.4753 Vimentin VIM EKLQEEMLQR NaN 0.77968 NaN Vimentin VIM ILLAELEQIK 0.7214 0.71178 1.3289 Vimentin VIM ILLAELEQLKGQGK 0.7299 NaN 2.7399 Vimentin VIM LGDLYEEEMR 0.52115 0.45107 0.90103 Vimentin VIM LQEEMLQR 0.87912 0.8627 1.8464 Vimentin VIM QDVDNASIAR 0.82023 0.89058 1.652 Vimentin VIM QVDQLTNDK 0.79102 0.72609 1.3024 Vimentin VIM RQVDQLTNDK 0.84806 0.70013 1.2391 Vimentin VIM VEVERDNLAEDIMR 0.58525 0.77955 1.0746 Female-lethal(2)D hom

WTAP EGNTTEDDFPSSPG NaN NaN NaN NGNK Female-lethal(2)D hom

WTAP LTNGPSNGSSSR NaN NaN NaN Female-lethal(2)D hom

WTAP QQLAQYQQQQSQAS 0.05126 NaN 0.67594 APSTSR Female-lethal(2)D hom

WTAP TSGSGFHR NaN NaN NaN μl μl Exactive pmol PrESTs PrESTs Exactive pmol/ Exactive pmol pmol pmol pmol pmol Protein (1 + 2) (3) pmol/μl 1 μl 2 pmol/μl 3 PrEST 1 PrEST 2 PrEST 3 Protein 1 Protein 2 3 0.021 0.0086 186.99624 129.566 51.550628 3.926921 2.720886 0.443335 0.654225 0.433791 0.527613 0.021 0.0086 186.99624 129.566 51.550628 3.926921 2.720886 0.443335 0.686936 0.486277 0.592163 0.021 0.0086 186.99624 129.566 51.550628 3.926921 2.720886 0.443335 0.695772 0.510189 0.582011 0.021 0.0086 186.99624 129.566 51.550628 3.926921 2.720886 0.443335 0.661608 0.58197 0.767502 0.006 0.0215 179.22945 128.578 56.812437 1.075377 0.771468 1.221467 1.7277 1.212825 1.596214 0.006 0.0215 179.22945 128.578 56.812437 1.075377 0.771468 1.221467 1.638874 1.094173 1.706268 0.006 0.0215 179.22945 128.578 56.812437 1.075377 0.771468 1.221467 1.835668 1.205342 2.025926 0.099 0.0421 222.86724 206.449 84.899424 22.06386 20.43845 3.574266 4.606713 4.212569 5.038285 0.099 0.0421 222.86724 206.449 84.899424 22.06386 20.43845 3.574266 1.659445 3.357629 #VALUE! 0.099 0.0421 222.86724 206.449 84.899424 22.06386 20.43845 3.574266 4.79602 4.697778 4.60008 0.099 0.0421 222.86724 206.449 84.899424 22.06386 20.43845 3.574266 2.796814 #VALUE! 1.702566 0.099 0.0421 222.86724 206.449 84.899424 22.06386 20.43845 3.574266 5.183241 5.508163 5.517951 0.099 0.0421 222.86724 206.449 84.899424 22.06386 20.43845 3.574266 3.17521 3.265043 4.617951 0.099 0.0421 222.86724 206.449 84.899424 22.06386 20.43845 3.574266 4.528386 3.641928 5.214854 0.099 0.0421 222.86724 206.449 84.899424 22.06386 20.43845 3.574266 4.224787 3.228458 4.567554 0.099 0.0421 222.86724 206.449 84.899424 22.06386 20.43845 3.574266 4.566777 4.97431 5.246665 0.297 0.4921 163.24925 124.502 55.082409 48.48503 36.97709 27.10605 39.41833 29.41602 44.7033 0.297 0.4921 163.24925 124.502 55.082409 48.48503 36.97709 27.10605 37.8799 29.99027 39.98956 0.297 0.4921 163.24925 124.502 55.082409 48.48503 36.97709 27.10605 #VALUE! 28.8303 #VALUE! 0.297 0.4921 163.24925 124.502 55.082409 48.48503 36.97709 27.10605 34.9771 26.31956 36.02123 0.297 0.4921 163.24925 124.502 55.082409 48.48503 36.97709 27.10605 35.38922 #VALUE! 74.26788 0.297 0.4921 163.24925 124.502 55.082409 48.48503 36.97709 27.10605 25.26797 16.67926 24.42337 0.297 0.4921 163.24925 124.502 55.082409 48.48503 36.97709 27.10605 42.62416 31.90014 50.04862 0.297 0.4921 163.24925 124.502 55.082409 48.48503 36.97709 27.10605 39.76887 32.93106 44.7792 0.297 0.4921 163.24925 124.502 55.082409 48.48503 36.97709 27.10605 38.35263 26.8487 35.30292 0.297 0.4921 163.24925 124.502 55.082409 48.48503 36.97709 27.10605 41.11821 25.88877 33.58711 0.297 0.4921 163.24925 124.502 55.082409 48.48503 36.97709 27.10605 28.37586 28.82549 29.12817 0.006 0.0028 178.53242 165.579 59.162252 1.071195 0.993474 0.165654 #VALUE! #VALUE! #VALUE! 0.006 0.0028 178.53242 165.579 59.162252 1.071195 0.993474 0.165654 #VALUE! #VALUE! #VALUE! 0.006 0.0028 178.53242 165.579 59.162252 1.071195 0.993474 0.165654 0.054909 #VALUE! 0.111972 0.006 0.0028 178.53242 165.579 59.162252 1.071195 0.993474 0.165654 #VALUE! #VALUE! #VALUE! Median CVs 12.33 Average CVs 18.39

indicates data missing or illegible when filed

To independently assess the precision of this step of absolute protein quantification, we compared the ratios determined from ‘limit tryptic peptides’ (those without internal arg or lys) to those determined from the longer versions of the peptide containing one or two missed tryptic cleavage sites. These peptides are very problematic for peptide standard based methods such as AQUA, but in our measurements very similar ratios were measured for such peptides. This shows that digestion proceeded identical for PrEST and endogenous protein (Table 1). Thus, far from introducing uncertainty, in the SILAC-PrEST approach these peptides can provide additional quantification information.

TABLE 1 Comparison of limit tryptic peptides and peptides with missed tryptic cleavage sites. Peptides with one or two miss cleavages as well as their ratios are depicted. The ratios of the two versions vary on average by 19%, which is in the normal range of variation of peptides derived from one protein. Gene Missed Ratio H/L CV Ratio H/L CV Ratio H/L CV Names Sequence Cleavages Mastermix1 (%) Mastermix2 (%) Mastermix3 (%) HSPA4 ELAILLGMLDPAEK 0 0.707 16.2 0.803 6.7 1.072 19.7 HSPA4 ELAILLGMLDPAEKDEK 1 0.562 0.730 1.418 HSPA4 EDQYDHLDAADMTK 0 0.220 9.2 0.132 25.8 0.558 35.6 HSPA4 NKEDQYDHLDAADMTK 1 0.193 0.191 0.933 ATP53 VLDSGAPIK 0 0.738 11.8 0.452 46.9 1.009 6.5 ATP5B VLDSGAPIKIPVGPETLGR 1 0.872 0.901 1.107 PPIB DKPLKDVIIADCGK 2 0.526 6.9 0.436 14.6 0.889 23.2 PPIB DVIIADCGK 0 0.580 0.354 1.237 FASN QQEQQVPILEK 0 0.627 18.6 1.040 13.9 FASN RQQEQQVPILEK 1 0.481 0.853 FEN1 LDPNKYPVPENWLHK 1 0.680 8.1 0.632 0.3 1.279 14.4 FEN1 YPVPENWLHK 0 0.607 0.629 1.043 SFN EKVETELQGVCDTVLGLLDSHLIK 1 0.442 6.3 0.389 6.4 1.188 1.2 SFN VETELQGVCDTVLGLLDSHLIK 0 0.483 0.426 1.168 SFN SNEEGSEEK 0 0.286 11.0 SFN SNEEGSEEKGPEVR 1 0.334 SEN YLAEVATGDDK 0 0.371 1.3 0.395 19.7 1.012 8.5 SFN YLAEVATGDDKK 1 0.364 0.299 1.142 TPR LESALTELEQLR 0 0.139 17.4 0.121 13.0 1.243 10.9 TPR LESALTELEQLRK 1 0.177 0.145 1.064 VCP DHFEEAMR 0 0.187 11.1 0.134 35.7 1.712 59.7 VCP RDHFEEAMR 1 0.218 0.224 0.696 VCP YEMFAQTLQQSR 0 0.169 4.4 0.146 6.6 1.584 37.1 VCP KYEMFAQTLQQSR 1 0.159 0.133 0.926 VIM QVDQLTNDK 0 0.620 8.5 0.813 2.3 VIM RQVDQLTNDK 1 0.699 0.788 VIM LQEEMLQR 0 0.868 3.9 0.834 65.0 VIM EKLQEEMLQR 1 0.821 0.309

To assess the degree of variability associated with both steps of the absolute quantification procedure, we repeated the entire workflow two more times, including PrEST quantification and master mix generation as well as measurement of cellular abundance of the target proteins. This analysis showed that the standard errors of the mean associated with all steps together are on average 24%. This value is excellent and to our knowledge the most accurate determination of cellular expression levels reported so far. Even more importantly, the errors of each of the step in the workflow for each of the proteins are immediately apparent from the individual CVs. Thus all protein expression level measurements can be classified and accepted or discarded according to the confidence of measurements. FIG. 4 displays typical examples of protein expression determination from the triplicate measurements. Comparing the peptide ratio spreads to the variability of the mean protein values revealed that the preparation of the master mix contributed the largest variability whereas errors due to SILAC ratio determination were somewhat lower. Automated preparation of the master mix could therefore lead to further improvements in the future.

Protein copy number determination in HeLa cells—Next we used the absolute values for protein amounts in our HeLa cell lysate to calculate the corresponding copy numbers in cells. HeLa cells numbers were determined automatically in a cell counter (see Experimental Procedures). Given the known amount of each PrEST and their SILAC ratios with respect to the endogenous proteins we determined the cellular copy numbers of 37 different proteins. Very high accuracy of absolute quantification to within a standard error of 25% was achieved for 30 of 37 proteins (Table 2).

TABLE 2 Protein Copy Numbers per HeLa cell Protein Names Gene Names Median SEM (%)* Master mix 1 Master mix 2 Master mix 3 14-3-3 protein sigma SFN 2,104,742 9.57 2,128,717 1,562,390 2,104,742 26S protease regulatory subunit 6A PSMC3 1,009,040 6.71 1,009,040 985,010 1,211,206 28S ribosomal protein S23, mitochondrial MRPS23 202,529 19.64 202,529 161,109 308,977 28S ribosomal protein S35, mitochondrial MRPS28 516,278 15.46 586,618 337,285 516,278 39S ribosomal protein L50, mitochondrial MRPL50 212,893 20.00 170,320 255,465 AFG3-like protein 2 AFG3L2 335,545 20.37 335,545 363,149 173,343 ATP synthase subunit beta, mitochondrial ATP5B 4,870,803 5.63 5,431,604 4,870,803 4,476,459 Carbonyl reductase [NADPH] 3 CBR3 101,019 63.47 101,019 60,715 498,397 Charged multivesicular body protein 6 CHMP6 133,137 24.10 154,916 61,839 133,137 COP9 signalosome complex subunit 5 COPS5 287,189 13.59 287,189 211,517 343,078 Cytochrome b5 reductase 4 CYB5R4 10,537 —** 10,537 — — Cytochrome b-c1 complex subunit 1, mitochondrial UQCRC1 1,032,315 8.96 1,032,315 808,601 1,099,145 Cytosolic acyl coenzyme A thioester hydrolase ACOT7 455,871 0.46 457,985 453,757 Endoplasmic reticulum lipid raft-associated protein 2 ERLIN2 135,785 18.00 218,008 127,563 135,785 Enoyl-CoA hydratase, mitochondrial ECHS1 2,162,058 15.20 2,705,599 1,574,948 2,162,058 Eukaryotic translation initiation factor 3 subunit 6 EIF3E 1,298,361 9.85 1,009,294 1,298,361 1,422,955 FACT complex subunit SSRP1 SSRP1 1,054,400 4.43 1,086,956 937,213 1,054,400 Fatty acid synthase FASN 3,361,337 13.11 4,093,238 2,575,577 3,361,337 Flap endonuclease 1 FEN1 2,215,232 6.87 2,215,232 2,220,140 1,789,805 Heat shock 70 kDa protein 4 HSPA4 1,719,164 11.22 1,855,515 1,258,240 1,719,164 Hepatocellular carcinoma-associated antigen 59 C9orf78 140,949 79.08 1,577,757 140,949 114,844 Mitogen-activated protein kinase scaffold protein 1 MAP2K1IP1 160,325 10.12 160,325 205,536 148,606 Nucleoprotein TPR TPR 306,362 14.08 343,837 208,601 306,362 Peptidyl-prolyl cis-trans isomerase B PPIB 11,155,435 17.40 14,871,040 8,035,119 11,155,435 Peroxiredoxin 6 PRDX6 8,815,042 3.13 9,010,737 8,118,496 8,815,042 Prefoldin subunit 1 PFDN1 358,511 22.03 383,937 358,511 171,199 Pre-mRNA-splicing regulator WTAP WTAP 72,199 — 72,199 — — Probable ATP-dependent RNA helicase DDX20 DDX20 268,121 — 268,121 — — Proto-oncogene c-Fos FOS 3,651 15.95 4,559 2,575 3,651 Purine nucleoside phosphorylase NP 1,618,680 10.11 1,832,987 1,284,556 1,618,680 Ras GTPase-activating-like protein IQGAP1 IQGAP1 1,322,762 15.42 1,667,348 963,468 1,322,762 SRA stem-loop-interacting RNA-binding protein, mitochondrial C14orf156 1,482,399 15.25 1,557,983 919,914 1,482,399 T-complex protein 1 subunit beta CCT2 4,352,706 29.38 8,283,044 3,162,779 4,352,706 THO complex subunit 1 THOC1 191,319 9.53 211,743 151,702 191,319 Transitional endoplasmic reticulum ATPase VCP 2,343,243 10.89 2,343,243 1,716,701 2,493,783 Vimentin VIM 20,600,599 8.73 20,600,599 17,557,991 23,805,318 Zinc finger protein 828 C13orf8 167,150 29.45 117,929 216,371 — *Standard error of the mean (SEM) for the three replicates in percent. **no valid data obtained

Cellular copy numbers are only known for very few proteins and it is therefore interesting to relate these copy numbers to the known functions of the proteins (Suppl. Table 3). The cytoskeletal protein vimentin forms intermediate filaments and was the most abundant protein with 20 million copies per cell. At the other extreme, the transcription factor and oncogene FOS is present in about 4,000 copies in our HeLa cell sample. As expected, proteins involved in cell signaling are generally expressed at lower values—as an example even the scaffolding factor mitogen-activated protein kinase scaffold protein 1 (MAP2K1IP1) is present at only 160,000 copies. However, ubiquitous signaling factors with a general chaperone-like role—such as 14-3-3 isoforms—are very highly expressed (14-3-3 sigma; 2.1 million copies). Two members of the mitochondrial ribosome have about 200,000 copies in this cell line (L23 and L5), whereas a third (L35) has about 500,000 (Note that not all ribosomal protein subunits have equal stoichiometry). The mitochondrial genome only encodes 13 genes therefore it is perhaps surprising that proteins involved in their translation are needed in such high copy numbers. A member of the respiratory chain, ATP5B, has about 5 million copies per HeLa cells—about five fold higher than PSMC3, a regulatory component of the proteasome. The T-complex is a member of a chaperone system and as expected it has a very high copy number (about 4 million). Fatty acid synthase, a classical enzyme, is expressed at 3.4 million copies, whereas another enzyme acyl coenzyme A thioester hydrolase (ACOT7) is expressed about seven-fold lower (450,000 copies). Such expression numbers could be interesting for modeling metabolic pathways. These are anecdotal examples but they illustrate that knowledge of the absolute expression levels of cellular proteins can contribute to the understanding of their roles in the cell.

SUPPLEMENTARY TABLE 3 corresponding UniProt link for the 43 proteins used for protein copy number determination. Protein Names Gene Names Function 14-3-3 protein sigma SFN http://www.uniprot.org/uniprot/P31947 26S protease regulatory subunit 6A PSMC3 http://www.uniprot.org/uniprot/P17980 28S ribosomal protein S23, mitochondrial MRPS23 http://www.uniprot.org/uniprot/Q9Y3D9 28S ribosomal protein S35, mitochondrial MRPS28 http://www.uniprot.org/uniprot/Q9Y2Q9 39S ribosomal protein L50, mitochondrial MRPL50 http://www.uniprot.org/uniprot/Q8N5N7 AFG3-like protein 2 AFG3L2 http://www.uniprot.org/uniprot/Q9Y4W6 ATP synthase subunit beta, mitochondrial ATP5B http://www.uniprot.org/uniprot/P06576 Carbonyl reductase [NADPH] 3 CBR3 http://www.uniprot.org/uniprot/O75828 Charged multivesicular body protein 6 CHMP6 http://www.uniprot.org/uniprot/Q96FZ7 COP9 signalosome complex subunit 5 COPS5 http://www.uniprot.org/uniprot/Q92905 Cytochrome b5 reductase 4 CYB5R4 http://www.uniprot.org/uniprot/Q7L1T6 Cytochrome b-c1 complex subunit 1, mitochondrial UQCRC1 http://www.uniprot.org/uniprot/P31930 Cytosolic acyl coenzyme A thioester hydrolase ACOT7 http://www.uniprot.org/uniprot/O00154 Endoplasmic reticulum lipid raft-associated protein 2 ERLIN2 http://www.uniprot.org/uniprot/O94905 Enoyl-CoA hydratase, mitochondrial ECHS1 http://www.uniprot.org/uniprot/P30084 Eukaryotic translation initiation factor 3 subunit 6 EIF3E http://www.uniprot.org/uniprot/P60228 FACT complex subunit SSRP1 SSRP1 http://www.uniprot.org/uniprot/Q08945 Fatty acid synthase FASN http://www.uniprot.org/uniprot/Q6PJJ3 Flap endonuclease 1 FEN1 http://www.uniprot.org/uniprot/P39748 Heat shock 70 kDa protein 4 HSPA4 http://www.uniprot.org/uniprot/P34932 Hepatocellular carcinoma-associated antigen 59 C9orf78 http://www.uniprot.org/uniprot/Q9NZ63 Mitogen-activated protein kinase scaffold protein 1 MAP2K1IP1 http://www.uniprot.org/uniprot/Q9UHA4 Nucleoprotein TPR TPR http://www.uniprot.org/uniprot/P12270 Peptidyl-prolyl cis-trans isomerase B PPIB http://www.uniprot.org/uniprot/P23284 Peroxiredoxin 6 PRDX6 http://www.uniprot.org/uniprot/P30041 Prefoldin subunit 1 PFDN1 http://www.uniprot.org/uniprot/O60925 Pre-mRNA-splicing regulator WTAP WTAP http://www.uniprot.org/uniprot/Q15007 Probable ATP-dependent RNA helicase DDX20 DDX20 http://www.uniprot.org/uniprot/Q9UHI6 Proto-oncogene c-Fos FOS http://www.uniprot.org/uniprot/P01100 Purine nucleoside phosphorylase NP http://www.uniprot.org/uniprot/P00491 Ras GTPase-activating-like protein IQGAP1 IQGAP1 http://www.uniprot.org/uniprot/P46940 SRA stem-loop-interacting RNA-binding protein, mitochondrial C14orf156 http://www.uniprot.org/uniprot/Q9GZT3 T-complex protein 1 subunit beta CCT2 http://www.uniprot.org/uniprot/P78371 THO complex subunit 1 THOC1 http://www.uniprot.org/uniprot/Q96FV9 Transitional endoplasmic reticulum ATPase VCP http://www.uniprot.org/uniprot/P55072 Vimentin VIM http://www.uniprot.org/uniprot/P08670 Zinc finger protein 828 C13orf8 http://www.uniprot.org/uniprot/Q96JM3

Absolute Quantification using heavy PrESTs—Above we used already expressed and purified PrESTs and quantified against heavy ABP protein and heavy SILAC-labeled cell lysate. While convenient to determine copy numbers in cell lines, in other applications it would be more appropriate to express heavy labeled PrESTs, which can then be mixed into any proteome of choice—including tissue and clinical body fluid samples. To apply our absolute quantification approach to non-labeled samples we expressed 28 of the PrESTs in heavy SILAC labeled E. coli, purified them and prepared a heavy master mix. To streamline quantification of PrEST levels, we developed an automated set up employing static nanoelectrospray (Advion NanoMate; see Example 1). As expected, spiking the heavy master mix into normal, non-SILAC labeled cells allowed equally straightforward quantification of the targeted proteins, with good correlation to the previous experiment (FIG. 7). Detailed information about the identification and quantification of the proteins is provided in Supplementary Table 4.

SUPPL. TABLE 4 All identification and quantification information of the  experiment in which heavy PrESTs were spiked into unlabeled HeLa lysate Protein Gene Ratio H/L Ratio H/L Ratio H/L Conc. μl PrESTs Protein Names Name Sequence Mastermix1 Mastermix2 Mastermix3 μg/μl (1 + 2) AFG3-like protein 2 AFG3L2 EQYLYTK NaN NaN NaN 0.779 0.01284 AFG3-like protein 2 AFG3L2 HFEQAIER NaN NaN 1.0384 0.779 0.01284 AFG3-like protein 2 AFG3L2 HLSDSINQK 0.98061 1.034 NaN 0.779 0.01284 AFG3-like protein 2 AFG3L2 LASLTPGFSGADVA 0.64771 0.68664 0.73842 0.779 0.01284 NVCNEAALIAAR AFG3-like protein 2 AFG3L2 TVAYHEAGHAVAGW 0.99036 0.88405 1.0667 0.779 0.01284 YLEHADPLLK AFG3-like protein 2 AFG3L2 VSEEIFFGR 0.88034 1.0026 0.087458 0.779 0.01284 ATP synthase subunit beta, ATP5B IMNVIGEPIDER 0.49547 0.38751 0.46817 0.359 0.25036 ATP synthase subunit beta, ATP5B IPVGPELTGR 0.67213 0.52947 0.5949 0.359 0.25036 ATP synthase subunit beta, ATP5B LVLEVAQHLGESTV 0.57216 0.53836 0.64057 0.359 0.25036 R ATP synthase subunit beta, ATP5B TIAMDGTEGLVR 0.52173 0.3056 0.41416 0.359 0.25036 ATP synthase subunit beta, ATP5B VLDSGAPIK 0.69347 0.569 0.60973 0.359 0.25036 ATP synthase subunit beta, ATP5B VLDSGAPIKIPVGP 0.71587 0.67498 NaN 0.359 0.25036 ETLGR Carbonyl reductase [NADPH CBR3 AFENCSEDLQER 3.7066 3.6554 2.7849 0.692 0.01445 Carbonyl reductase [NADPH CBR3 FHSETLTEGDLVDL 0.82772 0.54401 0.65383 0.692 0.01445 MK Carbonyl reductase [NADPH CBR3 VVNISSLQCLR NaN 0.61063 0.027081 0.692 0.01445 T-complex protein 1 subuni CCT2 HGINCFINR 0.96794 0.9253 0.80254 0.392 0.38219 T-complex protein 1 subuni CCT2 ILIANTGMDTDK 0.86038 0.52336 0.58541 0.392 0.38219 T-complex protein 1 subuni CCT2 LALVTGGEIASTFD 1.0899 0.99631 1.1095 0.392 0.38219 HPELVK T-complex protein 1 subuni CCT2 LIEEVMIGEDK 0.83525 0.94634 0.78472 0.392 0.38219 T-complex protein 1 subuni CCT2 VAEIEHAEK 1.1668 0.99657 1.1107 0.392 0.38219 T-complex protein 1 subuni CCT2 VAEIEHAEKEK 0.84991 0.99839 NaN 0.392 0.38219 COP9 signalosome complex COPS5 DHHYFK 0.72801 0.3227 0.37943 0.999 0.01001 COP9 signalosome complex COPS5 ISALALLK NaN NaN 0.23709 0.999 0.01001 Cytochrome b5 reductase 4 CYB5R4 QGHISPALLSEFLK NaN 12.575 13.552 0.867 0.01153 Cytochrome b5 reductase 4 CYB5R4 TEDDIIWR 11.628 12.424 12.299 0.867 0.01153 Probable ATP-dependent R

DDX20 VLISTDLTSR 0.59562 NaN NaN 0.705 0.01419 Enoyl-CoA hydratase, mitoc ECHS1 EGMTAFVEK 1.0475 NaN 0.02831 0.342 0.58542 Enoyl-CoA hydratase, mitoc ECHS1 ESVNAAFEMTLTEG 1.0239 NaN 0.98226 0.342 0.58542 SK Enoyl-CoA hydratase, mitoc ECHS1 ICPVETLVEEAIQC 0.77883 0.73163 0.73992 0.342 0.58542 AEK μl PrESTs NanoMate NanoMate NanoMate pmol PrEST pmol pmol pmol pmol pmol (3) pmol/μl pmol/μl pmol/μl 1 PrEST 2 PrEST 3 Protein 1 Protein 2 Protein 3 0.01284 18.253386 22.899283 0.2343735 0.294027 0 0.01284 18.253386 22.899283 0.2343735 0.294027 0 0 0.01284 18.253386 22.899283 0.2343735 0.294027 0 0.239008 0.284359 0.01284 18.253386 22.899283 0.2343735 0.294027 0 0.361849 0.428211 0 0.01284 18.253386 22.899283 0.2343735 0.294027 0 0.236655 0.332591 0 0.01284 18.253386 22.899283 0.2343735 0.294027 0 0.266231 0.293264 0 0.25036 23.934268 23.197529 19.970078 5.9921835 5.807733 4.999709 12.09394 14.98731 10.67926 0.25036 23.934268 23.197529 19.970078 5.9921835 5.807733 4.999709 8.915215 10.96896 8.404284 0.25036 23.934268 23.197529 19.970078 5.9921835 5.807733 4.999709 10.47292 10.78782 7.805093 0.25036 23.934268 23.197529 19.970078 5.9921835 5.807733 4.999709 11.48522 19.00436 12.07193 0.25036 23.934268 23.197529 19.970078 5.9921835 5.807733 4.999709 8.640869 10.20691 8.199873 0.25036 23.934268 23.197529 19.970078 5.9921835 5.807733 4.999709 8.370491 8.604304 0.01445 71.889794 33.419567 44.296682 1.0388075 0.482913 0.640087 0.280259 0.132109 0.229842 0.01445 71.889794 33.419567 44.296682 1.0388075 0.482913 0.640087 1.255023 0.887691 0.978981 0.01445 71.889794 33.419567 44.296682 1.0388075 0.482913 0.640087 0.790843 23.63602 0.38219 17.42877 12.336037 0 6.661102 4.71471 0 7.198856 5.874735 0.38219 17.42877 12.336037 0 6.672907 4.71471 0 12.75013 8.053689 0.38219 17.42877 12.336037 0 6.672907 4.71471 0 6.697622 4.2494 0.38219 17.42877 12.336037 0 6.672907 4.71471 0 7.051279 6.008143 0.38219 17.42877 12.336037 0 6.672907 4.71471 0 6.695874 4.244809 0.38219 17.42877 12.336037 0 6.672907 4.71471 0 6.683668 0.01001 14.240375 16.354906 0.1425462 0.163713 0 0.195802 0.507321 0 0.01001 14.240375 16.354906 0.1425462 0.163713 0 0 0.01153 21.356568 20.719244 0.2462412 0.238893 0 0.018997 0 0.01153 21.356568 20.719244 0.2462412 0.238893 0 0.021177 0.019228 0 0.01419 20.843513 13.238853 14.941964 0.295695 0.187859 0.212026 0.496574 0.58542 7.2168512 13.696164 7.5470525 4.224889 8.018008 4.418195 4.033307 156.5016 0.58542 7.2168512 13.696164 7.5470525 4.224889 8.018008 4.418195 4.126271 4.49799 0.58542 7.2168512 13.696164 7.5470525 4.224889 8.018008 4.418195 5.424661 10.9591 5.97118 Protein Gene Ratio H/L Ratio H/L Ratio H/L Conc. μl PrESTs Protein Names Name Sequence Mastermix1 Mastermix2 Mastermix3 μg/μl (1 + 2) Enoyl-CoA hydratase, mitoc ECHS1 IVVAMAK NaN NaN 1.063 0.342 0.58542 Enoyl-CoA hydratase, mitoc ECHS1 LFYSTFATDDR 1.5764 1.5022 1.6594 0.342 0.58542 Enoyl-CoA hydratase, mitoc ECHS1 LFYSTFATDDRK NaN 1.391 NaN 0.342 0.58542 Enoyl-CoA hydratase, mitoc ECHS1 SLAMEMVLTGDR 0.5035 0.63232 0.092059 0.342 0.58542 Eukaryotic translation initia EIF3F LGHVVMGNNAVSPY 0.11968 0.12455 0.048128 0.714 0.01401 QQVIEK Eukaryotic translation initia EIF3F LNMTPEEAER NaN 0.088999 NaN 0.714 0.01401 Eukaryotic translation initia EIF3F SQMLAMNIEK 0.096546 0.096998 0.081968 0.714 0.01401 Eukaryotic translation initia EIF3F WIVNLIR NaN 0.1375 0.082782 0.714 0.01401 Endoplasmic reticulum lipid ERLIN2 ADAECYTAMK 0.50502 2.1428 1.9075 0.186 0.05364 Endoplasmic reticulum lipid ERLIN2 DIPNMFMDSAGSVS NaN 0.36952 0.33782 0.186 0.05364 K Endoplasmic reticulum lipid ERLIN2 LSFGLEDEPLETAT 0.46716 0.51738 4.6718 0.186 0.05364 K Endoplasmic reticulum lipid ERLIN2 LTPEYLQLMK 0.76436 0.99457 0.95839 0.186 0.05364 Endoplasmic reticulum lipid ERLIN2 VAQVAEITYGQK NaN 3.8443 4.775 0.186 0.05364 Flap endonuclease 1 FEN1 EAHQLFLEPEVLDP 0.55946 0.53151 0.62173 0.883 0.07927 ESVELK Flap endonuclease 1 FEN1 HLTASEAK 0.57294 NaN NaN 0.883 0.07927 Flap endonuclease 1 FEN1 KLPIQEFHLSR NaN 0.50335 0.51855 0.883 0.07927 Flap endonuclease 1 FEN1 LDPNKYPVPENWLH 0.45538 0.53912 0.63616 0.883 0.07927 K Flap endonuclease 1 FEN1 LPIQEFHLSR NaN 0.56689 0.65691 0.883 0.07927 Flap endonuclease 1 FEN1 SIEEIVR 0.46634 0.2953 NaN 0.883 0.07927 Flap endonuclease 1 FEN1 VYAAATEDMDCLTF 0.25573 0.23882 0.2507 0.883 0.07927 GSPVLMR Flap endonuclease 1 FEN1 YPVPENWLHK 0.48958 0.15763 0.65879 0.883 0.07927 Heat shock 70 kDa protein 

HSPA4 EDQYDHLDAADMTK NaN NaN 0.64887 0.769 0.26011 Heat shock 70 kDa protein 

HSPA4 LNLQNK 2.3137 NaN 2.1319 0.769 0.26011 Heat shock 70 kDa protein 

HSPA4 NKEDQYDHLDAADM 0.76949 0.53896 0.54666 0.769 0.26011 TK Heat shock 70 kDa protein 

HSPA4 QSLTMDPVVK 0.50413 0.4421 0.54603 0.769 0.26011 Heat shock 70 kDa protein 

HSPA4 STNEAMEWMNNK 0.056431 0.045314 0.041474 0.769 0.26011 39S ribosomal protein L50, MRPL50 AYTPPEDLQSR 0.65073 NaN NaN 0.684 0.01462 39S ribosomal protein L50, MRPL50 LESYVK 1.131 NaN NaN  0.684 0.01462 28S ribosomal protein S23, MRPS23 ALLAEGVILR NaN 0.91056 0.4868 0.407 0.02459 28S ribosomal protein S23, MRPS23 LFVETGK NaN 1.0838 NaN 0.407 0.02459 28S ribosomal protein S23, MRPS23 LGETDEEK 0.93474 1.0182 1.0733 0.407 0.02459 μl PrESTs NanoMate NanoMate NanoMate pmol PrEST pmol pmol pmol pmol pmol (3) pmol/μl pmol/μl pmol/μl 1 PrEST 2 PrEST 3 Protein 1 Protein 2 Protein 3 0.58542 7.2168512 13.696164 7.5470525 4.224889 8.018008 4.418195 4.56346 0.58542 7.2168512 13.696164 7.5470525 4.224889 8.018008 4.418195 2.680087 5.337511 2.66526 0.58542 7.2168512 13.696164 7.5470525 4.224889 8.018008 4.418195 5.764204 0.58542 7.2168512 13.696164 7.5470525 4.224889 8.018008 4.418195 8.391041 12.6803 47.99309 0.01401 19.370034 20.330459 17.530494 0.2713742 0.28483 0.245602 2.267498 2.286871 5.103105 0.01401 19.370034 20.330459 17.530494 0.2713742 0.28483 0.245602 3.20037 0.01401 19.370034 20.330459 17.530494 0.2713742 0.28483 0.245602 2.810828 2.936449 2.996318 0.01401 19.370034 20.330459 17.530494 0.2713742 0.28483 0.245602 2.071489 2.966855 0.05364 24.33193 24.52011 22.095083 1.3051647 1.315259 1.18518 2.584382 0.613804 0.621326 0.05364 24.33193 24.52011 22.095083 1.3051647 1.315259 1.18518 3.559371 3.508319 0.05364 24.33193 24.52011 22.095083 1.3051647 1.315259 1.18518 0.279383 2.254215 0.253688 0.05364 24.33193 24.52011 22.095083 1.3051647 1.315259 1.18518 1.707526 1.32244 1.236637 0.05364 24.33193 24.52011 22.095083 1.3051647 1.315259 1.18518 0.342132 0.248205 0.07927 20.539053 20.564795 16.396092 1.6281308 1.630171 1.299718 2.910183 3.067057 2.090487 0.07927 20.539053 20.564795 16.396092 1.6281308 1.630171 1.299718 2.841713 0.07927 20.539053 20.564795 16.396092 1.6281308 1.630171 1.299718 3.238644 2.506447 0.07927 20.539053 20.564795 16.396092 1.6281308 1.630171 1.299718 3.575323 3.023763 2.043068 0.07927 20.539053 20.564795 16.396092 1.6281308 1.630171 1.299718 2.87564 1.978533 0.07927 20.539053 20.564795 16.396092 1.6281308 1.630171 1.299718 3.491296 5.52039 0.07927 20.539053 20.564795 16.396092 1.6281308 1.630171 1.299718 6.366601 6.825941 5.184357 0.07927 20.539053 20.564795 16.396092 1.6281308 1.630171 1.299718 3.325566 10.34176 1.972887 0.26011 32.693479 24.878715 0 8.503901 6.471203 9.973034 0.26011 32.693479 24.878715 0 8.503901 6.471203 0 3.035416 0.26011 32.693479 24.878715 0 8.503901 6.471203 0 15.77835 11.83771 0.26011 32.693479 24.878715 0 8.503901 6.471203 0 19.23524 11.85137 0.26011 32.693479 24.878715 0 8.503901 6.471203 0 187.6661 156.0303 0.01402 23.657325 15.986181 18.15397 0.3458701 0.233718 0.254519 0.531511 0.01402 23.657325 15.986181 18.15397 0.3458701 0.233718 0.254519 0.305809 0.02459 14.832962 15.409572 12.873057 0.3647425 0.378921 0.316548 0.416141 0.650264 0.02459 14.832962 15.409572 12.873057 0.3647425 0.378921 0.316548 0.349623 0.02459 14.832962 15.409572 12.873057 0.3647425 0.378921 0.316548 0.390207 0.372148 0.29493 Protein Gene Ratio H/L Ratio H/L Ratio H/L Conc. μl PrESTs Protein Names Name Sequence Mastermix1 Mastermix2 Mastermix3 μg/μl (1 + 2) 28S ribosomal protein S23, MRPS23 TQHGGSHVSR NaN 1.0302 NaN 0.407 0.02459 28S ribosomal protein S23, MRPS23 YTELQK NaN NaN 1.1676 0.407 0.02459 28S ribosomal protein S28, MRPS28 AGGFASALER NaN 0.51434 NaN 0.449 0.02229 28S ribosomal protein S28, MRPS28 HSELLQK 0.58177 NaN NaN 0.449 0.02229 Purine nucleoside phospho

NP ACVMMAQGR 0.50497 0.63931 0.56821 1.085 0.09389 Purine nucleoside phospho

NP DHINLPGFSGQNPL 0.95404 0.985 0.93693 1.065 0.09389 R Purine nucleoside phospho

NP FEVGDIMLIR 0.68963 0.70356 0.71547 1.065 0.09389 Purine nucleoside phospho

NP FHMYEGYPLWK 0.72381 0.90091 0.76466 1.065 0.09389 Purine nucleoside phospho

NP HRPQVAILCGSGLG NaN 0.85363 0.81538 1.065 0.09389 GLTDK Purine nucleoside phospho

NP LTQAQIFDYGEIPN 1.118 1.8464 1.8793 1.065 0.09389 FPR Purine nucleoside phospho

NP LVFGFLNGR 1.1247 0.30888 1.2496 1.065 0.09389 Purine nucleoside phospho

NP STVPGHAGR 1.0994 NaN 0.49901 1.065 0.09389 Purine nucleoside phospho

NP VFHLLGVDTLVVTN NaN 1.0676 1.0636 1.065 0.09389 AAGGLNPK Poly[ADP-ribose]polymera PARP4 AEGILLLVK NaN 0.58019 0.44594 0.579 0.01727 Prefoldin subunit 1 PFDN1 EAIHSQLLEK 0.76453 NaN NaN 0.441 0.09078 Prefoldin subunit 1 PFDN1 LADIQIEQLNR 1.6259 1.8677 1.9142 0.441 0.09078 Prefoldin subunit 1 PFDN1 MFILQSK 1.1344 NaN NaN 0.441 0.09078 Peroxiredoxin 6 PRDX6 DFTPVCTTELGR 0.56628 NaN 0.33394 0.714 0.56054 Peroxiredoxin 6 PRDX6 DINAYNCEEPTEK 0.61696 0.60109 0.5663 0.714 0.56054 Peroxiredoxin 6 PRDX6 ELAILLGMLDPAEK 0.57928 0.38932 0.38813 0.714 0.56054 Peroxiredoxin 6 PRDX6 FLAILLGMLDPAFK 0.56863 0.74994 0.21052 0.714 0.56054 DEK Peroxiredoxin 6 PRDX6 FHDFLGDSWGILFS NaN 0.57213 0.59897 0.714 0.56054 HPR Peroxiredoxin 6 PRDX6 GMPVTAR NaN 0.59916 0.51841 0.714 0.56054 Peroxiredoxin 6 PRDX6 LAPEFAK 0.80521 0.6713 0.74114 0.714 0.56054 Peroxiredoxin 6 PRDX6 LIALSIDSVEDHLA 0.74873 0.66939 0.62365 0.714 0.56054 WSK Peroxiredoxin 6 PRDX6 LPFPIIDDR 0.66166 0.68947 0.66519 0.714 0.56054 Peroxiredoxin 6 PRDX6 VVGVFGPDK 0.52861 0.25261 0.63133 0.714 0.56054 Peroxiredoxin 6 PRDX6 VVFVFGPDKK 0.84361 0.36625 0.63578 0.714 0.56054 26S protease regulatory sub PSMC3 AVCVEAGMIALR NaN 0.27688 0.067813 0.672 0.04466 26S protease regulatory sub PSMC3 GATELTHEDYMEGI NaN NaN NaN 0.672 0.04466 LEVQAK 26S protease regulatory sub PSMC3 MNVSPDVNYEELAR 0.67529 0.53568 0.63201 0.672 0.04466 FACT complex subunit SSRP SSRP1 ADVIQATGDAICIF 0.51416 0.56819 0.54776 0.587 0.05111 R μl PrESTs NanoMate NanoMate NanoMate pmol PrEST pmol pmol pmol pmol pmol (3) pmol/μl pmol/μl pmol/μl 1 PrEST 2 PrEST 3 Protein 1 Protein 2 Protein 3 0.02459 14.832962 15.409572 12.873057 0.3647425 0.378921 0.316548 0.367813 0.02459 14.832962 15.409572 12.873057 0.3647425 0.378921 0.316548 0.27111 0.02229 11.385909 11.881033 10.583484 0.2537919 0.264828 0.235906 0.514889 0.02229 11.385909 11.881033 10.583484 0.2537919 0.264828 0.235906 0.436241 0.09389 18.081184 27.459079 20.854165 1.6976424 2.578133 1.957998 3.361868 4.03268 3.445905 0.09389 18.081184 27.459079 20.854165 1.6976424 2.578133 1.957998 1.779425 2.617394 2.089801 0.09389 18.081184 27.459079 20.854165 1.6976424 2.578133 1.957998 2.461671 3.664411 2.736659 0.09389 18.081184 27.459079 20.854165 1.6976424 2.578133 1.957998 2.345425 2.861699 2.560612 0.09389 18.081184 27.459079 20.854165 1.6976424 2.578133 1.957998 3.020199 2.401331 0.09389 18.081184 27.459079 20.854165 1.6976424 2.578133 1.957998 1.518464 1.396302 1.041876 0.09389 18.081184 27.459079 20.854165 1.6976424 2.578133 1.957998 1.509418 8.346714 1.566899 0.09389 18.081184 27.459079 20.854165 1.6976424 2.578133 1.957998 1.544154 3.923764 0.09389 18.081184 27.459079 20.854165 1.6976424 2.578133 1.957998 2.414887 1.840915 0.01727 19.772134 22.931238 0.3414648 0.396022 0 0.682574 0 0.09078 16.010502 16.223891 1.4534334 1.472805 0 1.901081 0.09078 16.010502 16.223891 1.4534334 1.472805 0 0.893925 0.788566 0 0.09078 16.010502 16.223891 1.4534334 1.472805 0 1.281235 0.56054 19.343405 19.903505 12.598777 10.842752 11.15671 7.062119 19.14733 21.14787 0.56054 19.343405 19.903505 12.598777 10.842752 11.15671 7.062119 17.57448 18.5608 12.47063 0.56054 19.343405 19.903505 12.598777 10.842752 11.15671 7.062119 18.71764 28.65692 18.19524 0.56054 19.343405 19.903505 12.598777 10.842752 11.15671 7.062119 19.0682 14.8768 33.54607 0.56054 19.343405 19.903505 12.598777 10.842752 11.15671 7.062119 19.50031 11.79044 0.56054 19.343405 19.903505 12.598777 10.842752 11.15671 7.062119 18.62059 13.62265 0.56054 19.343405 19.903505 12.598777 10.842752 11.15671 7.062119 13.46574 16.61956 9.528724 0.56054 19.343405 19.903505 12.598777 10.842752 11.15671 7.062119 14.48153 16.66698 11.32385 0.56054 19.343405 19.903505 12.598777 10.842752 11.15671 7.062119 16.3872 16.18158 10.61669 0.56054 19.343405 19.903505 12.598777 10.842752 11.15671 7.062119 20.51182 44.16575 11.1861 0.56054 19.343405 19.903505 12.598777 10.842752 11.15671 7.062119 12.8528 30.46201 11.1078 0.04466 34.506036 32.565659 25.92524 1.5410396 1.454382 1.157821 5.252753 17.07374 0.04466 34.506036 32.565659 25.92524 1.5410396 1.454382 1.157821 0.04466 34.506036 32.565659 25.92524 1.5410396 1.454382 1.157821 2.282041 2.715021 1.831967 0.05111 18.671463 16.96063 13.294863 0.9542985 0.866858 0.6795 1.856034 1.525648 1.240508 Protein Gene Ratio H/L Ratio H/L Ratio H/L Conc. μl PrESTs Protein Names Name Sequence Mastermix1 Mastermix2 Mastermix3 μg/μl (1 + 2) FACT complex subunit SSRP SSRP1 ELQCLTPR NaN 0.48128 NaN 0.587 0.05111 FACT complex subunit SSRP SSRP1 IPYTTVLR 0.61437 0.57615 NaN 0.587 0.05111 THO complex subunit 1 THOC1 AVNNSNYGWR NaN NaN 0.96021 0.953 0.0105 THO complex subunit 1 THOC1 LWNLCPDNMEACK 0.17972 0.064595 0.05542 0.953 0.0105 THO complex subunit 1 THOC1 SLPEYLENMVIK NaN 0.063485 0.08477 0.953 0.0105 THO complex subunit 1 THOC1 TGEDEDEEDNDALL 1.9103 2.2196 1.3318 0.953 0.0105 K Nucleoprotein TPR TPR ILLSQTTGVAIPLH 2.5182 2.6647 2.8484 0.388 0.07732 ASSLDDVSLASTPK Nucleoprotein TPR TPR ITELQLK NaN NaN 2.4635 0.388 0.07732 Nucleoprotein TPR TPR LESALTELEQLR 2.3557 2.4601 2.3322 0.388 0.07732 Nucleoprotein TPR TPR LESALTELEQLRK NaN NaN 2.7043 0.388 0.07732 Nucleoprotein TPR TPR NIEELQQQNQR NaN 2.2529 2.0753 0.388 0.07732 Nucleoprotein TPR TPR QHQMQLVDSIVR 1.8112 NaN 1.9585 0.388 0.07732 Cytochrome b-c1 complex s UQCRC1 ADLTEYLSTHYK 0.21817 0.21768 0.25733 0.43 0.02326 Cytochrome b-c1 complex s UQCRC1 DVVFNYLHATAFQG 0.20672 0.22361 0.25427 0.43 0.02326 TPLAQAVEGPSENV R Cytochrome b-c1 complex s UQCRC1 MVLAAAGGVEHQQL 0.036719 0.16497 0.15532 0.43 0.02326 LDLAQK Vimentin VIM DNLAEDIMR 0.38208 0.423 0.40237 0.427 1.17023 Vimentin VIM EEAENTLQSFR 0.53572 0.51411 0.5603 0.427 1.17023 Vimentin VIM EKLQEEMLQR 0.35844 0.35626 0.35986 0.427 1.17023 Vimentin VIM ILLAELEQLK 0.60792 0.60021 0.62632 0.427 1.17023 Vimentin VIM ILLAELEQLKGQGK 0.45207 NaN NaN 0.427 1.17023 Vimentin VIM LGDLYEEEMR 0.41358 0.39183 0.30821 0.427 1.17023 Vimentin VIM LQEEMLQR 0.43217 0.27333 0.38427 0.427 1.17023 Vimentin VIM QDVDNASLAR 0.50789 0.50041 0.51027 0.427 1.17023 Vimentin VIM QVDQLTNDK 0.57376 0.52999 0.5087 0.427 1.17023 Vimentin VIM RQVDQLTNDK 0.5666 0.51939 0.6736 0.427 1.17023 μl PrESTs NanoMate NanoMate NanoMate pmol PrEST pmol pmol pmol pmol pmol (3) pmol/μl pmol/μl pmol/μl 1 PrEST 2 PrEST 3 Protein 1 Protein 2 Protein 3 0.05111 18.671463 16.96063 13.294863 0.9542985 0.866858 0.6795 1.801151 0.05111 18.671463 16.96063 13.294863 0.9542985 0.866858 0.6795 1.553296 1.50457 0.0105 26.314913 19.802314 20.98021 0.2763066 0.207924 0.220292 0.229421 0.0105 26.314913 19.802314 20.98021 0.2763066 0.207924 0.220292 1.537428 3.218892 3.974959 0.0105 26.314913 19.802314 20.98021 0.2763066 0.207924 0.220292 3.275172 2.598705 0.0105 26.314913 19.802314 20.98021 0.2763066 0.207924 0.220292 0.14464 0.093676 0.165409 0.07732 16.099976 16.486454 15.642843 1.2448502 1.274733 1.209505 0.494341 0.478378 0.424626 0.07732 16.099976 16.486454 15.642843 1.2448502 1.274733 1.209505 0.49097 0.07732 16.099976 16.486454 15.642843 1.2448502 1.274733 1.209505 0.528442 0.518163 0.518611 0.07732 16.099976 16.486454 15.642843 1.2448502 1.274733 1.209505 0.447252 0.07732 16.099976 16.486454 15.642843 1.2448502 1.274733 1.209505 0.565819 0.58281 0.07732 16.099976 16.486454 15.642843 1.2448502 1.274733 1.209505 0.687307 0.617567 0.02326 20.444076 15.780782 0 0.475529 0.667061 0 2.184533 1.426421 0.02326 20.444076 15.780782 0 0.475529 0.667061 0 2.126601 1.443587 0.02326 20.444076 15.780782 0 0.475529 0.667061 0 2.882519 2.363256 1.17023 14.055391 18.074527 15.696811 16.44804 21.15135 18.36888 43.04868 50.0032 45.65171 1.17023 14.055391 18.074527 15.696811 16.44804 21.15135 18.36888 30.70268 41.14169 32.78401 1.17023 14.055391 18.074527 15.696811 16.44804 21.15135 18.36888 45.88785 59.37055 51.04452 1.17023 14.055391 18.074527 15.696811 16.44804 21.15135 18.36888 27.05626 35.23992 29.32827 1.17023 14.055391 18.074527 15.696811 16.44804 21.15135 18.36888 36.38384 1.17023 14.055391 18.074527 15.696811 16.44804 21.15135 18.36888 39.76991 53.98094 59.59858 1.17023 14.055391 18.074527 15.696811 16.44804 21.15135 18.36888 38.05919 77.38394 47.80201 1.17023 14.055391 18.074527 15.696811 16.44804 21.15135 18.36888 32.38504 42.26805 35.99835 1.17023 14.055391 18.074527 15.696811 16.44804 21.15135 18.36888 28.66711 39.90897 36.10945 1.17023 14.055391 18.074527 15.696811 16.44804 21.15135 18.36888 29.02937 40.72345 27.26971

indicates data missing or illegible when filed

SUPPLEMENTARY TABLE 4b Copy Copy Copy Gene Mastermix 1 Mastermix 2 Mastermix 3 Number Number Number Name Protein Name (pmol) (pmol) (pmol) 1 2 3 Median RSD (%) AFG3L2 AFG3-like protein 2 0.252619235 0.312927491 0 152,131 152,131 ATP5B ATP synthase subunit beta, mitocl

9.694065359 10.87839063 8.404284127 5,837,904 6,551,121 5,061,179 5,837,904 12.81 AYTL2 Lysophosphatidylcholine — — — acyltrans

C1orf65 Uncharacterized protein C1orf65 — — — CBR3 Carbonyl reductase [NADPH]3 0.482912745 0.790843465 0.97898086 290,817 476,817 589,556 476,257 33.35 CCT2 T-complex protein 1 subunit 0 6.874450414 5.87473498 4,139,892 3,537,849 3,838,879 11.09 beta COPS5 COP9 signalosome 0.195802466 0.507321377 0 117,915 305,516 211,716 62.66 complex subun

CYB5R4 Cytochrome b5 reductase 4 0.021176576 0.019112892 0 12,753 11,510 12,131 7.24 DDX20 Probable ATP-dependent 0.49657408 — — 299,044 299,044 RNA hel

ECHS1 Enoyl-CoA hydratase, 4.126271164 8.361653639 5.234584905 2,484,899 5,035,506 3,152,341 3,152,341 37.18 mitochondri

EIF3E Eukaryotic translation 2.539162992 2.611659997 2.996318259 1,529,120 1,572,779 1,804,425 1,572,779 9.05 initiation fa

ERLIN2 Endoplasmic reticulum 1.707526217 0.613803749 0.621326468 1,028,296 369,641 374,172 374,172 64.16 lipid raft-a FEN1 Flap endonuclease 1 3.408430951 3.238643686 2.066777383 2,052,605 2,052,605 HSPA4 Heat shock 70 kDa 0 19.23524295 11.83771012 11,583,736 7,128,837 9,356,286 33.67 protein 4 MLKL Mixed lineage kinase — — — domain-like MRPL50 39S ribosomal protein 0.418660011 — — 252,123 252,123 L50, mitocl

MRPS23 28S ribosomal protein 0.390207475 0.369980833 0.294930107 234,988 177,611 206,300 19.67 S23, mitocl

MRPS28 28S ribosomal protein 0.436240972 0.441351419 0.271110383 262,710 265,788 163,267 262,710 25.29 S35, mitocl

NP Purine nucleoside 1.779424739 2.940949047 2.401331339 1,071,595 1,771,081 1,446,116 1,446,116 24.48 phosphorylase PARP4 Poly [ADP-ribose] — 0.682573768 0 411,056 411,056 polymerase 4 PFDN1 Prefoldin subunit 1 1.281235376 0.788566037 0 771,578 771,578 PRDX6 Peroxiredoxin 6 17.57448193 18.59069261 11.79043804 10,583,602 11,195579 7,100,369 10,583,602 22.95 PSMC3 26S protease regulatory 2.282041145 3.983887093 9.452851115 1,374,278 2,399,153 5,692,641 2,399,153 71.51 subunit 6 SSRP1 FACT complex subunit 1.704665071 1.52567761 1.240507611 1,026,573 918,767 747,051 918,767 15.71 SSRP1 THOC1 THO complex subunit 1 0.841034292 3.218891514 1.414062794 506,483 851,569 679,026 35.94 TPR Nucleoprotein 0.528441724 0.518162926 0.504790509 318,235 312,045 303,992 312,045 2.29 TPR UQCRC1 Cytochrome b-c1 complex 0 2.184533321 1.443587459 1,315,557 869,349 1,092453 28.88 subunit VIM Vimentin 34.38444006 42.26804731 36.10945422 20,706,797 25,454,417 21,745,625 21,745,625 11.03

indicates data missing or illegible when filed

SUPPLEMENTARY TABLE 4c forward experiment (light PrESTs) reverse experiment (heavy PrESTs) Gene Copy Copy Copy RSD Copy Copy Copy RSD RSD Name Protein Name Number 1 Number 2 Number 3 Median (%) Number 1 Number 2 Number 3 Median (%)  (%) AFG3L2 AFG3-like protein 2 369,737 412,509 165,983 369,737 41.68 152,131 152,131 58.96924 ATP5B ATP synthase subunit beta, 5,672,473 4,376,424 4,511,967 4,511,967 14.68 5,837,904 6,745,143 5,350,929 5,837,904 11.84 18.11769 mitocl

AYTL2 Lysophosphatidylcholine acyltransferase 1 C1orf65 Uncharacterized protein C1orf65 CBR3 Carbonyl reductase [NADPH]3 79,823 61,399 322,454 79,823 94.26 290,925 476,434 589,556 476,434 33.33 100.8334 CCT2 T-complex protein 1 subunit 7,447,762 2,757,533 4,479,130 4,479,130 48.47 4,139,892 3,537,849 3,838,870 11.09 10.8856 beta COPS5 COP9 signalosome complex 323,791 284,218 435,937 323,791 22.62 117,915 292,058 205,487 60.27 31.61052 subun

CYB5R4 Cytochrome b5 reductase 4 16,205 10,180 9,515 10,180 30.8  12,753 7,618 10,185 35.65 0.037884 DDX20 Probable ATP-dependent 242,403 184,529 213,466 19.17 299,044 299,044 23.61427 RNA heli

ECHS1 Enoyl-CoA hydratase, 2,965,394 1,723,133 2,105,336 2,105,336 28.1  2,484,899 5,276,677 3,157,087 3,157,087 40.03 28.26456 mitochondri

EIF3E Eukaryotic translation 1,067,627 599,306 1,253,469 1,067,627 34.63 1,529,120 1,531,715 1,870,428 1,531,751 11.94 25.24945 initiation fa ERLIN2 Endoplasmic reticulum lipid 206,262 148,785 149,867 149,867 19.53 1,028,296 395,520 308,967 395,520 67.99 63.69902 raft-a

FEN1 Flap endonuclease 1 2,373,346 2,019,699 1,563,785 2,019,699 20.42 2,052,605 2,052,605 1.142762 HSPA4 Heat shock 70 kDa protein 4 2,146,713 1,499,858 1,646,549 1,646,549 19.22 11,287,790 7,120,698 9,204,244 32.01 98.50151 MLKL Mixed lineage kinase 128,711 100,891 114,801 17.14 domain-like MRPL50 39S ribosomal protein L50, 177,937 250,001 194,935 194,935 18.14 252,123 252,123 18.09073 mitoch

MRPS23 28S ribosomal protein S23, 223,198 203,672 282,020 223,198 17.26 234,988 162,252 198,620 25.89 8.240033 mitoch

MRPS28 28S ribosomal protein S35, 473,409 284,783 422,825 422,825 24.8  262,710 128,718 149,148 149,148 40.06 67.66706 mitoch

NP Purine nucleoside 2,101,680 1,357,920 1,555,814 1,555,814 23.04 1,071,595 1,785,222 1,476,153 1,476,153 24.78 3.715642 phosphorylase PARP4 Poly [ADP-ribose] 60,775 67,168 63,971  7.07 413,808 413,808 103.5509 polymerase 4 PFDN1 Prefoldin subunit 1 476,849 523,643 243,332 476,849 36.22 771,578 771,578 33.3868 PRDX6 Peroxiredoxin 6 8,881,373 8,377,838 8,781,079 8,781,079  3.07 10,583,602 9,447,065 6,349,191 9,447,065 24.92 5.166992 PSMC3 26S protease regulatory 1,062,048 950,200 1,192,875 1,062,048 11.37 1,374,278 2,514,900 5,692,641 2,514,900 70.06 57.44122 subunit 6 SSRP1 FACT complex subunit SSRP1 1,095,695 1,022,209 1,209,724 1,095,695  8.52 1,026,573 911,155 747,051 911,155 15.70 13.00441 THOC1 THO complex subunit 1 239,173 184,576 204,962 204,962 13.16 506,483 851,569 679,026 35.94 75.84125 TPR Nucleoprotein TPR 397,408 278,736 357,637 357,637 17.53 318,235 315,936 303,992 315,936  2.45 8.755396 UQCRC1 Cytochrome b-c1 complex 1,022,450 713,318 1,025,854 1,022,450 19.5  1,312,073 888,889 1,100,481 27.19 5.198108 subunit VIM Vimentin 22,974,646 17,376,010 22,886,339 22,886,339 15.22 20,706,797 25,128,776 21,745,625 21,745,625 10.26 3.614478

indicates data missing or illegible when filed

Absolute quantification in single experiments—We also wished to develop a variation on the SILAC-PrEST strategy to quantify single protein target. In this case, the two experimental steps involved in absolute protein quantification can be collapsed into one as outlined schematically in FIG. 6A. A precisely known amount of the ABP solubility tag is mixed into cell lysate together with the labeled PrEST. LC-MS/MS analysis of the sample then provides SILAC ratios of light ABP solubility tag to labeled PrEST ABP peptides. These ratios accurately quantify the amount of PrEST that was used. The same LC MS data also contain the ratios of labeled PrEST peptides to the unlabeled endogenous protein counterpart. Together, these ratios quantify the absolute amount of endogenous protein in a single experiment. Note that triple-SILAC labeling is not required in this approach because the ratios are determined against different regions of the PrEST construct, namely the common ABP solubility tag region (for quantifying the PrEST) and the protein specific PrEST region (for quantifying the endogenous protein).

This single-plex method for quantification was performed for three different HeLa proteins in which the SILAC-labeled cell lysate and SILAC-labeled ABP was quantified against unlabeled PrESTs. As shown in FIG. 6C, consistent values were obtained in these measurements based on triplicate experiments. The absolute levels generally agreed well with the copy numbers determined independently in the multiplexed PrEST-SILAC experiment described above (maximum difference between the means of 40%), validating both approaches.

Enzyme-linked immunosorbent assay—ELISA is a standard method in biochemical research to determine absolute amounts, or at least to reproducibly determine protein levels. We therefore compared the SILAC-PrEST method to this established technology. When performing the ELISA assay for Stratifin (14-3-3 σ) under typical conditions—filtered cell lysate and phosphate buffered saline (PBS) as recommended by the manufacturer—the ELISA recorded less than 20% of the amount quantified by MS. (Note that there is no interference by 14-3-3 isoforms because these peptides are different.) The recommendation of the manufacturer was PBS could not solubilize the pellet. The solubility was increased by adding the nonionic detergent NP-40, which was able to dissolve most of the sample pellet. Adding a low concentration of sodium dodecyl sulfate (SDS), an anionic detergent further improvement significantly increased measured protein amount (FIG. 8B). Still the absolute amounts were underestimated two-fold compared to mass spectrometry analysis, presumably because the FASP protocol enables complete solubilization by the use of 4% sodium dodecyl sulfate.

We also investigated the levels of the transcription factor and proto-oncogene FOS by ELISA, the lowest abundance protein quantified in our mix. Here solubilization did not appear to be an issue and we received excellent agreement between quantitative values determined by MS and by ELISA using different buffer conditions (FIG. 8A).

Example 3 Absolute Quantification Using Mouse PrESTs

Experimental procedure—The mouse PrESTs fused with a N-terminal His-tag were expressed in an auxotrophic E. coli strain using minimal media, supplemented with isotope labeled ¹³C₆ ¹⁵N₂-Lysine (Lys8) and ¹³C₆ ¹⁵N₄-Arginine (Arg10) (Cambridge Isotopes Laboratories) to obtain ‘heavy’ labeled proteins. The bacteria were harvested by centrifugation, lysed in 7M guanidinium chloride, 47 mM Na₂HPO₄, 2.65 mM NaH₂PO₄, 10 mM Tris HCL, 300 mM NaCl, 10 mM beta-mercaptoethanol, pH 8.0 and the His-fusion PrESTs were enriched on a Cobalt Talon column (Clontech) and eluted in 6 M Urea, 50 mM NaH₂PO₄, 100 mM NaCl, 30 mM Acetic acid, 70 mM Na-acetate pH 5 (29).

Blood samples were drawn from mice into tubes containing heparin. The blood was centrifuged twice at 70 g and each time the supernatant, the platelet rich plasma (PRP), was retained. Apyrase and prostacyclin (PGI₂) were added to the PRP to inhibit platelet aggregation. The sample was centrifuged and the pellet was washed twice with 1 ml of Tyrode's buffer (without Ca²⁺, containing BSA, apyrase and PGI₂). Eventually the pellet was resuspended in 300-400 μl Tyrode's buffer and incubated for 30 min at 37° C. A standard hematologic analysis was performed using the Hemavet 950 (Drew Scientific Inc.) to count platelets.

The isolated platelets were lysed in 4% SDS, 100 mM Tris pH 8.5, 100 mM DTT, boiled for 5 min at 95° C. and the purified PrESTs were added to the lysate in the appropriate amount. The samples were prepared in accordance with the previously described FASP method (30). Peptides were collected by centrifugation and eluted with water. Peptides were desalted on C18 empore stages tips and eluted in buffer B (80% acetonitrile, 0.5% acetic acid), organic solvent was removed by speed-vacing and the sample was resolved in A* (2% acetonitrile, 0.5% acetic acid). The peptides were loaded without prefractionation on an in-house packed 20 cm column (75 μm inner diameter) packed with 1.8 μm C18 resin (Dr. Maisch GmbH) and separated using an EASY-nLC 1000 (Thermo Fisher Scientific) on a 200 min 2-25% buffer B gradient. The separated peptides were sprayed via a nanoelectrospray ion source (Proxeon Biosystems) to a Q Exactive mass spectrometer (Thermo Fisher Scientific). The mass spectrometer acquired survey scans and the top 10 most abundant ions were sequentially fragmented with higher-energy collisional dissociation and MS/MS scans acquired. Raw data was analyzed using the Max Quant software as described in Example 1 except that the data was searched against the mouse IPI database version 3.68 containing 56,743 entries.

Results—To further broaden the approach to other species we designed PrESTs targeting mouse proteins. PrESTs were designed to span over a 125-200 amino acids region, yielding many tryptic peptides and including numerous peptides that were observed in the mass spectrometer in previous measurements. For each target protein we designed two PrESTs to cover different regions of the proteins and to ensure quantification precision. We designed PrESTs to measure the expression levels of Integrin beta 3 and its co-activators Talin 1 and Kindlin 3 in mouse platelets. The activation of the heterodimer Integrin αIIbβ3 (shifting from a low-affinity state to an high affinity state) plays an essential role in platelet adhesion and aggregation (31). Mice deficient of Kindlin 3 suffer from severe bleeding and die within several days. We determined expression levels of Integrin beta 3, Talin 1 and Kindlin 3 in wild-type mice (Kind3^(+/+)), Kind3^(+/in), Kind3^(n/n) and Kind3^(n/−). ‘n’ indicates an insertion of a neomycin cassette into an intron of the gene, affecting splicing of Kindlin 3. To further elucidate functionality of Integrin activation we wished to measure the stoichiometry of Integrin beta 3, Talin 1 and Kindlin 3 in the wildtype mice.

Integrin beta 3 and its co-activators are highly abundant proteins in platelets and Itgb3 has on average 300,000 copies per cell, while its co-activators Talin 1 has 470,000 copies and Kindlin 3 has on average 430,000 copies per platelet (Table 3, FIG. 9 a). We measured copy numbers of the target proteins in the different mice in duplicates and using two different PrESTs. The difference between platelets samples was on average 20%, whereas the difference between PrESTs is 22%. For the Kindlin 3 calculation we only considered one PrESTs since this targets the region of biological interest—the C-terminus of Kindlin 3 interacts to the cytoplasmic tail of Integrin beta 3.

Besides the copies per cell we also observed the decrease of the expression level of Kindlin 3 in the different knock-outs (FIG. 9 b). In comparison to the wild-type mice Kindlin 3 diminished as expected to 50% in the Kind3^(+/n) mice, to 15% in Kind3^(n/n) mice and to 6% Kind3^(n/−) mice and the trend is in agreement with observations of Moser et al. (32).

TABLE 3 Copy numbers per platelet. The absolute amounts of the proteins of interest were measured each using two PrESTs in two mice samples. Integrin beta 3 Talin 1 Kindlin 3 % Kind3 +/+ 345,000 531,000 433,000 100 Kind3 +/n 306,000 445,000 242,000 55 Kind3 n/n 313,000 490,000 68,000 15 Kind3 n/− 268,000 409,000 26,000 6

Using the absolute amount the stoichiometry of the three proteins (Table 2) in the wild-type mice was determined to be 1:1.5:1.3 and this stoichiometry information helps to further understand the binding of co-activators and the activation of integrins.

TABLE 4 Stoichiometry of the protein calculated in wild-type mice using the absolute expression levels. Integrin beta 3 Talin 1 Kindlin 3 Copy number 345,000 531,000 433,000 Stoichiometry 1 1.5 1.3

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1. A method of determining the absolute amount of a target polypeptide in a sample, said method comprising the following steps: (a) adding (aa) a fusion polypeptide to said sample, said fusion polypeptide comprising (i) at least one tag sequence and (ii) a subsequence of the target polypeptide; and (ab) a known absolute amount of a tag polypeptide comprising or consisting of said tag sequence according to (aa) to said sample, wherein said fusion polypeptide on the one hand is mass-altered as compared to said target polypeptide and said tag polypeptide on the other hand, for example, said fusion polypeptide on the one hand and said target polypeptide and said tag polypeptide on the other hand are differently isotope labeled; (b) performing proteolytic digestion of the mixture obtained in step (a); (c) subjecting the result of proteolytic digestion of step (b), optionally after chromatography, to mass spectrometric analysis; and (d) determining the absolute amount of said target polypeptide from (i) the peak intensities in the mass spectrum acquired in step (c) of said fusion polypeptide, said tag polypeptide and said target polypeptide and (ii) said known absolute amount of said tag polypeptide.
 2. A method of creating a quantitative standard, said method comprising the following steps: (a) providing one or a plurality of fusion polypeptides, the one fusion polypeptide or each of said fusion polypeptides, respectively, comprising (i) at least one tag sequence and (ii) a subsequence of a target polypeptide to be quantitatively determined, wherein, to the extent said plurality of fusion polypeptides is provided, all fusion polypeptides share at least one tag sequence, thereby obtaining the standard; (b) determining the absolute amounts of said fusion polypeptide(s) by (ba) adding to the one fusion polypeptide or to one of said fusion polypeptides at a time, respectively, a known amount of a tag polypeptide comprising or consisting of the tag sequence comprised in the one fusion polypeptide or shared among the fusion polypeptides, respectively, according to (a), wherein said fusion polypeptide is mass-altered as compared to said tag polypeptide, for example, said fusion polypeptide and said tag polypeptide are differently isotope labeled, (bb) performing proteolytic digestion of the mixture of one fusion polypeptide and said tag polypeptide obtained in step (ba); (bc) subjecting of the result of proteolytic digestion of step (bb), optionally after chromatography, to mass spectrometric analysis; and (bd) determining the absolute amount of said one fusion polypeptide from (i) the peak intensities in the mass spectrum of fusion polypeptide and tag polypeptide and (ii) said known amount of said tag polypeptide, thereby obtaining the absolute amount of the one fusion polypeptide or of one of said plurality of fusion polypeptides at a time, respectively.
 3. A method of determining the absolute amount of one or more target polypeptides in a sample, said method comprising the following steps: (a) adding the quantitative standard as defined in claim 2(a) to said sample; (b) performing proteolytic digestion of the mixture obtained in step (b); (c) subjecting the result of proteolytic digestion of step (c), optionally after chromatography, to mass spectrometric analysis; and (d) determining the absolute amounts of the target polypeptide(s) from (i) the peak intensities in the mass spectrum acquired in step (d) of fusion polypeptide(s) and target polypeptide(s) and (ii) the known absolute amount(s) of said fusion polypeptide(s) wherein said fusion polypeptide(s) is/are mass-altered as compared to said target polypeptide(s), for example, said one or more target polypeptides are differently isotope labeled as compared to said fusion polypeptides.
 4. The method of claim 1, wherein one or two tags are present in said fusion polypeptide(s), said tag(s) being selected from a purification tag and a solubility tag.
 5. The method of claim 1, wherein said adding is effected prior to said proteolytic digestion.
 6. The method of claim 2, wherein between two and 500 fusion polypeptides are used.
 7. The method of claim 1, wherein a solubility tag is present in each of said fusion polypeptides.
 8. The method of claim 1, wherein said subsequence of a polypeptide (a) consists of 15 to 205 amino acids; (b) comprises a proteotypic peptide; and/or (c) is selected to have minimal sequence identity to other proteins, excludes signal peptides and/or excludes sequences from transmembrane spanning regions.
 9. A fusion polypeptide for the quantification of a target polypeptide by mass spectroscopy, wherein: said fusion polypeptide consists of 35-455 amino acid residues and comprises (i) a target region, which is a fragment of the target polypeptide, and (ii) a tag region, which is not a fragment of the target polypeptide, said target region consists of 15-205 amino acid residues and comprises at least two signature regions; said tag region consists of 20-250 amino acid residues and comprises at least two signature regions; each signature region has the structure Y-Z-X₄₋₂₈-Y-Z, wherein all Y:s are selected from one of (i)-(iv), wherein (i) is R or K, (ii) is Y, F, W or L, (iii) is E and (iv) is D and each X and each Z are independently any amino acid residue, provided that the Z:s are not P if the Y:s are selected from (i)-(iii); and each signature region comprises at least one amino acid residue comprising a heavy isotope.
 10. A fusion polypeptide according to claim 9, wherein said tag region corresponds to a solubility tag or a fragment thereof, said solubility tag being selected from Maltose-binding protein (MBP), Glutathione-S-transferase (GST), Thioredoxin (Trx), N-Utilization substance (NusA), Small ubiquitin-modifier (SUMO), a Solubility-enhancing tag (SET), a Disulfide forming protein C (DsbC), Seventeen kilodalton protein (Skp), Phage T7 protein kinase (T7PK), Protein G B1 domain (GB1), Protein A IgG ZZ repeat domain (ZZ) and Albumin Binding Protein (ABP).
 11. A fusion polypeptide according to claim 9, wherein said tag region consists of or comprises the sequence set forth in SEQ ID NO:
 1. 12. A fusion polypeptide according to claim 9, wherein the Y:s are selected from R and K.
 13. The method according to claim 1, wherein said fusion polypeptide(s) consist(s) of 35-455 amino acid residues and comprise(s) (i) a target region, which is a fragment of the target polypeptide, and (ii) a tag region, which is not a fragment of the target polypeptide, said target region consists of 15-205 amino acid residues and comprises at least two signature regions; said tag region consists of 20-250 amino acid residues and comprises at least two signature regions; each signature region has the structure Y-Z-X₄₋₂₈-Y-Z, wherein all Y:s are selected from one of (i)-(iv), wherein (i) is R or K, (ii) is Y, F, W or L, (iii) is E and (iv) is D and each X and each Z are independently any amino acid residue, provided that the Z:s are not P if the Y:s are selected from (i)-(iii); and each signature region comprises at least one amino acid residue comprising a heavy isotope.
 14. A kit comprising: (a) at least one fusion polypeptide according to claim 9; and (b) (i) a second polypeptide comprising or consisting of the amino acid sequence of the tag region as defined in claim 9, said second polypeptide being differently isotope labeled compared to said tag region as defined in claim 9; or (ii) a proteolytic enzyme, such as trypsin, chymotrypsin, Lys-C, Glu-C or Asp-N.
 15. Use of a quantitative standard as defined in claim 2 as a reference in a target polypeptide quantification.
 16. The method of claim 3, further comprising performing the method of claim 2 before adding.
 17. The method of claim 2, wherein one or two tags are present in said fusion polypeptide(s), said tag(s) being selected from a purification tag and a solubility tag.
 18. The method of claim 3, wherein one or two tags are present in said fusion polypeptide(s), said tag(s) being selected from a purification tag and a solubility tag.
 19. The method of claim 3, wherein between two and 500 fusion polypeptides are used.
 20. The method of claim 2, wherein a solubility tag is present in each of said fusion polypeptides.
 21. The method of claim 3, wherein a solubility tag is present in each of said fusion polypeptides.
 22. The method of claim 2, wherein said subsequence of a polypeptide (a) consists of 15 to 205 amino acids; (b) comprises a proteotypic peptide; and/or (c) is selected to have minimal sequence identity to other proteins, excludes signal peptides and/or excludes sequences from transmembrane spanning regions.
 23. The method according to claim 2, wherein said fusion polypeptide(s) consist(s) of 35-455 amino acid residues and comprise(s) (i) a target region, which is a fragment of the target polypeptide, and (ii) a tag region, which is not a fragment of the target polypeptide, said target region consists of 15-205 amino acid residues and comprises at least two signature regions; said tag region consists of 20-250 amino acid residues and comprises at least two signature regions; each signature region has the structure Y-Z-X₄₋₂₈-Y-Z, wherein all Y:s are selected from one of (i)-(iv), wherein (i) is R or K, (ii) is Y, F, W or L, (iii) is E and (iv) is D and each X and each Z are independently any amino acid residue, provided that the Z:s are not P if the Y:s are selected from (i)-(iii); and each signature region comprises at least one amino acid residue comprising a heavy isotope.
 24. Use of a fusion polypeptide according to claim 9 as a reference in a target polypeptide quantification. 